Dry powder formulations of dnase i

ABSTRACT

DNase I formulations for pulmonary administration and, more particularly, but not exclusively, a dry powder formulation comprising, as an active ingredient, human DNase I, methods, dry powder inhalation devices and systems for the therapeutic use thereof are provided.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to DNase Iformulations for pulmonary administration and, more particularly, butnot exclusively, to a dry powder formulation comprising, as an activeingredient, human DNase I, methods, dry powder inhalation devices andsystems for the therapeutic use thereof.

Human DNase I is a member of the mammalian DNase I family (EC 3.1.21.1).DNase I belongs to the class of Mg²⁺ and Ca²⁺ dependent endonucleases,whose hydrolytic activity depends on the presence of bivalent metals.Magnesium ion is involved in electrophilic catalysis of thephosphodiester bond cleavage, whereas Ca²⁺ maintains optimal enzymeconformation. DNase I cleaves DNA preferentially at phosphodiesterlinkages adjacent to a pyrimidine nucleotide, yielding5′-phosphate-terminated polynucleotides with a free hydroxyl group onposition 3′, on average producing tetranucleotides. It acts onsingle-stranded DNA, double-stranded DNA, and chromatin.

The principal therapeutic use of human DNase has been to reduce theviscoelasticity of pulmonary secretions (including mucus) in suchdiseases as pneumonia and cystic fibrosis (CF), thereby aiding in theclearing of respiratory airways. Mucus also contributes to the morbidityof chronic bronchitis, asthmatic bronchitis, bronchiectasis, emphysema,acute and chronic sinusitis, and even the common cold. DNase I iseffective in reducing the viscoelasticity of pulmonary secretions andfluids by hydrolyzing high-molecular-weight DNA present in pulmonarysecretions and fluids. DNase has also been proposed for non-pulmonarydisorders, for example, treatment of male infertility and uterinedisorders (see US 2007/0259367), inhibition of metastatic growth (seeU.S. Pat. No. 7,612,032) and for treatment of sepsis and viral,bacterial, fungal and protozoan infections.

DNA encoding human DNase I was isolated and sequenced, and expressed inrecombinant host cells, thereby enabling the production of human DNasein commercially useful quantities. Recombinant human DNase (rhDNase)(e.g. dornase alfa; Pulmozyme®, Genentech., CA), expressed in Chinesehamster ovary (CHO) cells, has been found to be clinically effective forCF.

Recombinant human DNase (rhDNase) (e.g. dornase alfa; Pulmozyme®,Genentech., CA) was approved for clinical use by the FDA in 1994, andadditional human clinical trials with recombinant human DNase I arecurrently in progress for CF (see NCT01155752; NCT0017998; NCT00117208and NCT00204685) and other chronic, respiratory diseases such asatelectasis (see NCT01095276 and NCT00671323) and Sjogren's Syndrome. Ingeneral, safety and efficacy of the rhDNase has been demonstrated.Pulmozyme® is provided as a liquid protein formulation ready for use innebulizer systems.

In addition to nebulizer systems, pulmonary administration of drugs andother pharmaceuticals can be accomplished by provision of a dry powderformulation for inhalation by means of suitable inhalers known as drypowder inhalers (DPIs).

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided an inhalable dry powder formulation comprising a humanDNase I protein and particles of a physiologically acceptablepharmacologically-inert solid carrier.

According to an aspect of some embodiments of the present inventionthere is provided a dry powder inhaler device, comprising the inhalabledry powder formulation comprising a human DNase I protein and particlesof a physiologically acceptable pharmacologically-inert solid carrier ameans for introducing the inhalable dry powder formulation into theairways of a subject by inhalation.

According to some embodiments of the present invention the device is asingle dose or a multidose inhaler.

According to some embodiments of the present invention the device ispre-metered or device-metered.

According to some embodiments of the present invention the human DNase Iprotein is a recombinant human DNase I protein.

According to some embodiments of the present invention the human DNase Iprotein is a plant-expressed human DNase I protein.

According to some embodiments of the present invention the human DNase Iprotein comprises an N-terminal Glycine residue.

According to some embodiments of the present invention the human DNase Iprotein comprises the amino acid sequence as set forth in SEQ ID NO: 6.

According to some embodiments of the present invention the human DNase Iprotein comprises the amino acid sequence as set forth in SEQ ID NO:5.

According to some embodiments of the present invention the human DNase Iprotein has at least one core xylose and at least one core α-(1,3)fucose.

According to some embodiments of the present invention the human DNase Iprotein has reduced susceptibility to actin inhibition of endonucleaseactivity as compared with that of mammalian cell expressed humanrecombinant DNase I.

According to some embodiments of the present invention the carrier isselected from the group consisting of (a) at least one crystalline sugarselected from the group consisting of glucose, arabinose, maltose,saccharose, dextrose, and lactose; and (b) at least one polyalcoholselected from the group consisting of mannitol, maltitol, lactitol, andsorbitol.

According to some embodiments of the present invention the carrier is ina form of finely divided particles having a mass median diameter (MMD)in the range of 0.5 to 10 microns.

According to some embodiments of the present invention the carrier is ina form of finely divided particles having a mass median diameter (MMD)in the range of 1.0 to 6.0 microns.

According to some embodiments of the present invention the carrier is ina form of coarse particles having a mass diameter of 50-500 microns.

According to some embodiments of the present invention the coarseparticles have a mass diameter of 150 microns to 400 microns.

According to some embodiments of the present invention the carriercomprises a mixture of coarse particles having a mass diameter of 150microns to 400 micron and finely divided particles having a MMD in therange of 0.5-10 microns.

According to some embodiments of the present invention the dry powderformulation further comprises, as an active ingredient, a magnesiumsalt.

According to some embodiments of the present invention the dry powderformulation further comprises, as an active ingredient, an agent forinhibiting formation of G actin and/or enhancing formation of F actin.

According to some embodiments of the present invention the dry powderformulation further comprises one or more additive materials selectedfrom the group consisting of an amino acid, a water soluble surfaceactive agent, a lubricant, and a glidant.

According to some embodiments of the present invention the human DNase Iprotein is at least 90-95% pure human DNase I protein.

According to some embodiments of the present invention the inhalablepharmaceutical composition further comprising plantbeta-acetylhexosaminidase enzyme protein. In some embodiments the plantbeta-acetylhexosaminidase enzyme protein is inactivatedbeta-acetylhexosaminidase enzyme protein.

In some embodiments the beta-acetylhexosaminidase enzyme protein is heatinactivated beta-acetylhexosaminidase enzyme protein.

According to some embodiments of the present invention the human DNase Iis in association with diketopiperazine.

According to some embodiments of the present invention thediketopiperazine is selected from the group consisting of succinyldiketopiperazine, glutaryl diketopiperazine, maleyl diketopiperazine,and fumaryl diketopiperazine or a pharmaceutically acceptable saltthereof.

According to an aspect of some embodiments of the present inventionthere is provided a method of reducing extraceullular DNA in a subjectin need thereof, the method comprising administering to the subject inneed thereof an effective amount of the inhalable dry powder formulationcomprising human DNase I of the invention.

According to some embodiments of the present invention the subject issuffering from a disease or condition selected from the group consistingof male infertility, metastatic cancer, a viral, bacterial, fungal orprotozoan infection, sepsis, atherosclerosis, diabetes, delayed typehypersensitivity and a uterine disorder.

According to an aspect of some embodiments of the present inventionthere is provided a method for reducing DNA in a pulmonary secretion orfluid of a subject in need thereof, the method comprising administeringto the subject in need thereof an effective amount of the inhalable drypowder formulation comprising human DNase I of the invention.

According to an aspect of some embodiments of the present inventionthere is provided a method for the prevention and/or treatment of apulmonary disease or condition associated with excess DNA in a pulmonarysecretion in a subject in need thereof, the method comprisingadministering to a subject in need thereof an effective amount of theinhalable dry powder formulation comprising human DNase I of theinvention.

According to some embodiments of the present invention the subject issuffering from a respiratory disease selected from the group consistingof acute or chronic bronchopulmonary disease, atelectasis due totracheal or bronchial impaction, and complications of tracheostomy.

According to some embodiments of the present invention the acute orchronic bronchopulmonary disease is selected from the group consistingof infectious pneumonia, bronchitis or tracheobronchitis,bronchiectasis, cystic fibrosis, asthma, chronic obstructive pulmonarydisease (COPD), TB or fungal infections.

According to some embodiments of the present invention the effectiveamount of the dry powder formulation is a single dose of 0.1 to 25 mgDNase I, administered daily.

According to some embodiments of the present invention the effectiveamount of the dry powder formulation is a single dose of 0.5 to 15 mgDNase I, administered daily.

According to some embodiments of the present invention the effectiveamount of the dry powder formulation is a single dose of 1.0 to 10 mgDNase I, administered daily.

According to some embodiments of the present invention the effectiveamount of the dry powder formulation is a single dose of 2.0 to 5 mgDNase I, administered daily.

According to some embodiments of the present invention the effectiveamount of the dry powder formulation is a single dose of 2.0-3.0 mgDNase I, administered daily.

According to some embodiments of the present invention the effectiveamount of the dry powder formulation is a plurality of doses, each dosecomprising 1.0-3.0 mg DNase, the doses administered at least twice, 2-3times, 2-4 times or 2-6 times daily.

According to some embodiments of the present invention the effectiveamount of the dry powder formulation is a plurality of doses, each dosecomprising 1.0-3.0 mg DNase, the doses administered once every 36 hours,once every 36-48 hours, once every 36-72 hours, once every 2-3 days,once every 2-4 days, once every 2-5 days, or once every week.

According to some embodiments of the present invention the effectiveamount of the dry powder formulation is a plurality of doses, each dosecomprising 1.0-3.0 mg DNase, the doses administered once every 36 hours,once every 36-48 hours, once every 36-72 hours, once every 2-3 days,once every 2-4 days, once every 2-5 days, or once every week.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is an amino acid sequence alignment of the plant recombinanthuman DNase I (prhDNase I) encoded by the nucleic acid of the invention[SEQ ID NO: 1, sequence (b)], and native human DNase I protein[(GenBank: NM_(—)005223, sequence (a)] (SEQ ID NO: 2), including thenative signal leader peptide (highlighted in red, SEQ ID NO: 3). TheArabidopsis ABPI endoplasmic reticulum targeting signal peptide (SEQ IDNO: 4) is highlighted in green;

FIG. 2 is a graph showing the results of targeting expression ofrecombinant human DNase I to different plant organelles. rhDNase I wasexpressed in whole tobacco plants and targeted for secretion (to theapoplast) via an N-terminal ER targeting signal peptide (APO), targetedto the vacuole, via an N-terminal ER targeting signal and a C-terminalvacuolar targeting signal peptide (VAC) and targeted to the endoplasmicreticulum via an N-terminal ER targeting signal peptide and a C-terminalER retention signal peptide (ER). Activity of rhDNase I in the cells ofthe tobacco plant was monitored by immunoreactivity (ELISA) andaccording to catalytic activity (DNA-methyl green assay), adjusted foramount of biomass in sample and expressed as phrDNase I relative toexpression in apoplast-targeted cells (100%). Note the significantlygreater yield when the rhDNase I is targeted to the apoplast;

FIG. 3 shows an SDS-PAGE gel illustrating the size and purity ofplant-expressed rh DNase I. 1.25 μg, 2.5 μg, 5 μg and 10 μg ofcommercial human DNase (Pulmozyme®) (lanes 1-4, respectively) orpurified prh DNase I (lanes 6-9, respectively) were separated on 15%Tris-Glycine SDS-PAGE, stained with Coomassie blue, and analyzed incomparison to molecular weight standards (lanes 5 and 10). Note thesingle, predominant band of the plant-expressed enzyme migratingslightly but discernibly faster than that of commercial human DNase(Pulmozyme®), and the absence of detectable protein impurities;

FIG. 4 shows an SDS-PAGE gel illustrating the size, purity and antigenicidentity of plant-expressed rh DNase I. 10 ng, 50 ng, 100 ng and 200 ngof commercial human DNase (Pulmozyme®) (lanes 1-4, respectively) orpurified prh DNase I (lanes 6-9, respectively) were separated on 15%Tris-Glycine SDS-PAGE, transferred to nitrocellulose membrane, probedwith whole antiserum primary antibody obtained from rabbits immunizedagainst Pulmozyme®, and visualized with secondary goat anti-rabbit IgGHRP-conjugated antibody. Note the single, predominant band of theimmunoreactive, plant-expressed enzyme migrating and reacting similarlyto that of commercial human DNase (Pulmozyme®), and the absence ofdetectable protein impurities;

FIGS. 5A-5D shows an IEF gel and capillary electropherogram of humanDNase (Pulmozyme®) and purified, plant expressed rh DNase I. FIG. 5Ashows the resolution of 6 μg of commercial human DNase (Pulmozyme®)(lane 1) or 6 μg of purified, plant expressed rh DNase I (lanes 2-3) onan isoelectric focusing gel. FIG. 5B shows the resolution of 8 μg ofcommercial human DNase (Pulmozyme®) (lane 3) or 8 μg of purified, plantexpressed rh DNase I from a different line of BY2 cells expressing theprhDNase I (lane 2) on an isoelectric focusing gel. Note that Pulmozyme®resolves into multiple bands with isoelectric points (pI) between pI 3.5and pI 4.5 in a pH gradient of pH 3 to pH 7, while in both of thepurified, plant expressed rh DNase I preparations the DNase I resolvedat an isoelectric point (pI) between pI 4.2 and pI 4.5, in two majorbands and one minor band. Electrophoresis conditions included 100 mV-1hour, 200 mV-1 hour and 500 mV-1.5 hours. Lanes 4 (FIG. 5A) and lane 1(FIG. 5B) contain protein standards for comparison. FIG. 5D is anelectropherogram of image capillary isoelectric focusing (scale=pI3.3-6.1) analysis of purified, plant expressed rh DNase I, showing theresolution of the prh DNase I as observed in the IEF gel [one major peak(3) at pI 4.41 and two minor peaks (4 and 5) at pI 4.27 and 4.21). 1 and2 are pI markers 5.85 and 3.59. FIG. 5C is an electropherogram of theblank scale with pI markers 1 and 2;

FIGS. 6A-6B show the molecular mass analysis of purified, plantexpressed rh DNase I by mass spectrometry using 2.5 micrograms of thepurified prh DNase I using a matrix-assisted laser desorption ionizationtime-of-flight (MALDI-ToF) mass spectrometer, with sinapinic acid as amatrix. FIG. 6A showing the entire spectrum from 20000 to 180000 m/z andFIG. 6B showing an enlarged segment of the prh-DNaseI peak at about32000 m/z;

FIG. 7 is a figure representing a putative amino acid sequence of thepurified, plant expressed rh DNase I (SEQ ID NO: 5), derived from thesequences of overlapping peptides produced by partial proteolyticdigestion (see SEQ ID NOs: 17-276) and separated and analyzed on RP-HPLCand mass spectrometry. Unconfirmed amino acids are marked in red,glycosylation sites are underlined and the N-terminal Glycine residue ismarked in green;

FIGS. 8A-8C represent analysis of the glycan structure of the purified,plant expressed rh DNase I, as determined by exoglucosidase digestionand NP-HPLC analysis of the resulting de-glysosylated polypeptides. FIG.8A is an NP-HPLC profile of total glycans released following PNGase Adigestion of the glycosylated prhDNase I, derived from three separateexemplary batches of prhDNase I. The numbers above the discrete peakscorrespond to specific glycan structures illustrated in FIG. 8B. FIG. 8Bis a chart showing individual glycan structures released from prhDNase Iby PNGase A digestion, and their relative abundance (in percentages)from total released glycans, for each of the three exemplary batchestested. Note the predominance of two main glycan peaks representing ahigh percentage of glycan structures (over 80%) containing mannose3-β-(1,2) xylose-α-(1,3) fucose [Fc(3)M3X] and/or mannose 4-α-(1,2)xylose. FIG. 8C shows an NP-HPLC profile of total glycans released fromprhDNase I using PNGase A endoglycosidase (top) compared to the profileof N-linked glycans released from Pulmozyme® using PNGase Fendoglycosidase (specific to high mannose glycans) showing a multitudeof glycan variations. The major peaks are annotated with correspondingglycan structures. Note the wide variation in glycosylation patternfound in Pulmozyme®, due to the abundance of bi- and tri-antennaryglycans, and sialic acid residues.

FIGS. 9A-9D are chromatograms of RP-HPLC of purified, plant expressed rhDNase I, analyzed at 214 nm (FIG. 9A, 9B) and 280 nm (FIG. 9C, 9D),indicating a preparation of the prh DNase I having less than 7%impurities (93.77% pure measured at 214 nm, and 93.62% pure measured at280 nm). Insets (FIGS. 9B and 9D) are expanded views of the prh DNase Ipeak at 214 nm and 280 nm, respectively;

FIGS. 10A-10B are graphs representing substrate inhibition kinetic plotsof purified prh DNase I (open circles ◯) and commercial human DNase(Pulmozyme®) (closed circles ), using the DNaseAlert™ fluorometricsubstrate detected at 535 nm and 565 nm. FIG. 10A is a plot of theinitial velocity as a function of substrate concentration. Note thesuperior kinetics (greater V_(max), lower K_(M), e.g. higher specificactivity) of the purified prh DNase I. FIG. 10B is a double reciprocalplot of initial velocity as a function of substrate concentration. K_(M)and V_(max) values were calculated using the following linear regressionequations (plotted at substrate concentration of 2-15 μM) and R² values:prh DNase I: y=41.377x+1.8693, R²=0.9894; Pulmozyme®: y=120.17x+4.8316,R²=0.9921, indicating substrate-inhibition-like kinetics for both prhDNase I and Pulmozyme®;

FIG. 11 is an activity plot of purified, plant expressed rh DNase I(open circles ◯) and commercial human DNase (Pulmozyme®) (closed circles), illustrating resistance to actin inhibition of DNase I activity. ΔOD(620 nm) is plotted as a function of G-actin concentration, using theMethyl Green substrate, to yield IC₅₀ values. Note the reducedsusceptibility to G-actin inhibition of the purified prh DNase I,compared to that of commercial human DNase (Pulmozyme®);

FIGS. 12A-D are histograms illustrating the effect of increasingconcentrations of DNase I on the elastic modulus of sputum from CysticFibrosis patients, determined using time-sweep measurements with arheomoter (HAAKE RheoStress I, Thermo Fisher Scientific GmBH, Germany).FIG. 12A represents a comparison of the effect of purified, plantexpressed rh DNase I (dark bars) and commercial human DNase (Pulmozyme®)(hatched bars) on the elastic modulus of CF patient sputa, measured at2, 10 and 20 μg DNase I/gr sputum. DNA content of the sputum was 4.66 μgDNA/gr sputum. Each value represents at least 2 determinations measuredfrom each sputum sample. FIGS. 12B-12D show the effect of prhDNase I onthe elastic modulus of sputa collected from individual patients. Notethe pronounced, superior and consistent, dose-dependent reduction ofsputum elastic modulus following incubation with prh DNase I, ascompared to the Pulmozyme®;

FIGS. 13A-D are histograms illustrating the effect of increasingconcentrations of DNase I on the viscous modulus of sputum from CysticFibrosis patients, measured using time-sweep technique with a rheomoter(HAAKE RheoStress I, Thermo Fisher Scientific GmBH, Germany). FIG. 13Arepresents a comparison of the effect of purified, plant expressed rhDNase I (dark bars) and commercial human DNase (Pulmozyme®) (hatchedbars) on the viscous modulus of CF patient sputa, measured at 2, 10 and20 μg DNase I/gr sputum. DNA content of the sputum was 4.66 μg DNA/grsputum. Each value represents at least 2 determinations measured fromeach sputum sample. FIGS. 13B-13D shows the effect of prhDNase I on theviscous modulus of sputa collected from individual patients. Note thepronounced, superior and consistent, dose-dependent reduction of sputumviscous modulus following incubation with prh DNase I, as compared tothe Pulmozyme®;

FIGS. 14A and 14B are histograms representing the effect of prhDNase Ion the rheological properties of sputum from Cystic Fibrosis patients,measured using time sweep technique, expressed as percent changerheological properties (elastic modulus, FIG. 14A and viscous modulus,FIG. 14B) compared to an untreated sample. FIG. 14A shows the percentchange of elastic modulus of the sputa of 4 individual patients (blackbars=untreated control samples; hatched bars=2 μg prhDNase I; graybars=20 μg prhDNase I). FIG. 14B shows the percent change of viscousmodulus of the sputa of 4 individual patients (black bars=untreatedcontrol samples; hatched bars=2 μg prhDNase I; gray bars=20 μg prhDNaseI). Note the consistent pronounced reduction in rheological propertiesfollowing incubation of the sputa with prh DNase I;

FIG. 15 is a histogram illustrating the synergic effect of purified,plant expressed rh DNase I on the reduction of CF sputum elastic modulusby Magnesium (MgCl₂) (0, 25, 50 and 100 mM), measured using time sweeptechnique, with rheomoter (HAAKE RheoStress I, Thermo Fisher ScientificGmBH, Germany). Elastic modulus of the sputum was determined atindicated concentrations of Mg²⁺ (mM MgCl₂) in the presence(cross-hatched bars) or absence (dark bars, buffer only) of prh DNase I(2 μg prh DNase I/gr sputum). Each value represents at least 2determinations. Note the synergic reduction of sputum elastic modulusfollowing incubation with the prh DNase I and Magnesium, as compared toMagnesium alone;

FIG. 16 is a histogram illustrating the effect of Magnesium salts onDNase I reduction of CF sputum viscous modulus. FIG. 16 shows thesynergic effect of purified, plant expressed rh DNase I on the reductionof CF sputum viscous modulus by Magnesium (MgCl₂) (0, 25, 50 and 100mM), measured with a rheomoter (HAAKE RheoStress I, Thermo FisherScientific GmBH, Germany). Viscous modulus of the sputum was determinedat the indicated concentrations of Mg²⁺ (mM MgCl₂) in the presence(cross hatched bars) or absence (grey bars, buffer only) of prh DNase I(2 μg DNase I/gr sputum). Each value represents at least 2determinations. Note the synergic reduction of sputum viscous modulusfollowing incubation with the prh DNase I and Magnesium, as compared toMagnesium alone;

FIG. 17 is a graph illustrating the effect of different Magnesium saltson the DNase catalytic activity of purified, plant expressed rh DNase I.Catalytic activity of rh DNase I was measured in the presence ofincreasing concentrations (0.5-100 mM) of Magnesium chloride (MgCl₂,closed circles ) or Magnesium sulphate (MgSO₄, open diamonds ⋄), usingthe methyl green substrate and expressed as the change in absorbance at630 nm (Δ OD 630 nm) after 3 hours incubation with the enzyme. Note thatneither magnesium salt inhibited rh DNase I activity up to 50 mM, andthat rh DNase I activity was only slightly impaired by MgSO₄ at 100 mM.

FIGS. 18A-18D are histograms illustrating the effect of increasingconcentrations of DNase I on the rheological properties of sputum fromCystic Fibrosis patients, determined using the stress sweep techniquewith a rheomoter (HAAKE RheoStress I, Thermo Fisher Scientific GmBH,Germany), expressed as the stress (Pa) at cross-over of elastic andviscous modules (phase angle=45°). FIGS. 18A-18D represents a comparisonof the effect of purified, plant expressed rh DNase I (dark bars) andcommercial human DNase (Pulmozyme®) (hatched bars) on the rheologicalproperties of four individual CF patient sputa, measured at 0.2, 2 and20 μg DNase I/gr sputum. DNA content of the sputum was 3.09 mg DNA/grsputum, 3.15 mg DNA/gr sputum, 8.36 mg DNA/gr sputum and 3.89 mg DNA/grsputum (FIGS. 18A-18D, respective). Each value represents at least 2determinations measured from each sputum sample. Note the pronounced andconsistent, dose-dependent reduction in cross-over stress valuesfollowing incubation with prh DNase I;

FIGS. 19A to 19D are histograms representing the effect of prhDNase Iand Pulmozyme® on the rheological properties of sputum from fourdifferent Cystic Fibrosis patients (19A-19D), measured using stresssweep technique, expressed as the stress (Pa) at cross-over of elasticand viscous modules (phase angle=45°). DNA content of the sputum was2.16 mg DNA/gr sputum, 2.63 mg DNA/gr sputum, 3.45 mg DNA/gr sputum and4.17 mg DNA/gr sputum (FIGS. 19A-19D, respective) Black bars=prhDNase I;hatched bars=Pulmozyme®. DNase I concentration (0, 2 or 20 μg DNase I/grsputum). Note the consistent pronounced reduction in rheologicalproperties following incubation of the sputa with prh DNase I,significantly outperforming Pulmozyme® in 3 out of the four patients;

FIGS. 20A-20C are histograms illustrating the effect of Magnesium saltsand DNase I on the rheological properties of CF sputum samples fromthree different Cystic Fibrosis patients, measured using the stresssweep technique with a rheomoter (HAAKE RheoStress I, Thermo FisherScientific GmBH, Germany), expressed as the stress (Pa) at cross-over ofelastic and viscous modules (phase angle=45°). FIG. 20A—patient A (1.84mgDNA/g sputum), FIG. 20B—patient B (3.46 mgDNA/g sputum) and FIG. 20C(2.39 mg DNA/g sputum). Dark columns are measurements without Magnesium,grey columns are represent measurement with 100 mM MgSO₄, measured at 0(control) 2 and 20 μg DNase I/gr sputum. Each value represents at least2 determinations measured from each sputum sample. Note the pronouncedand synergistic disruption of sputum elastic structure followingincubation with prh DNase I and MgSO₄.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to drypowder formulations of human DNase I as an active ingredient, suitablefor administration by inhalation by means of a dry powder inhaler (DPI),processes for the preparation of such a formulation, and methods ofusing such a formulation for reducing extracellular DNA in a subject inneed thereof and for the prevention and/or treatment of a wide range ofconditions including respiratory disorders such as cystic fibrosis (CF),asthma and chronic obstructive pulmonary disease (COPD), as well asother, non-respiratory disorders.

It is to be understood that the invention is not necessarily limited inits application to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

Clinically approved, commercially available recombinant human DNase I(rh DNase I) (Dornase Alpha®, Pulmozyme®; Genentech) is supplied as aliquid protein formulation for use in pulmonary administration byinhalation using a nebulizer. Such a liquid formulation is unsuitablefor use in other forms and systems for pulmonary administration ofdrugs, such as dry powder inhalation. The present inventors have deviseda dry powder formulation comprising human DNase I, suitable forpulmonary administration and capable of providing catalytically activehuman DNase I for nucleolytic digestion of extracellular DNA found insecretions, fluids and tissues of the airways, as well as fluids,secretions and tissues accessible via systemic circulation.

Surprisingly, such dry powder formulations, typically noted for theirefficacy in delivering pharmaceutical compositions to the respiratorylining and alveolar cells, have been found effective for providingcatalytically active human DNase I protein to the sputum and otherpulmonary secretions, resulting in reduction in the DNA content andimproving the rheological properties of the sputum and other secretions.Yet further, when comprising a plant expressed recombinant human DNaseI, the efficacy of the dry powder formulation of the invention intreating pulmonary secretions can be enhanced due to the surprisinglyfavorable kinetic properties and resistance of the prh DNase I to actininhibition, thereby enhancing the therapeutic value of the human DNase Iwhen provided in such a dry powder formulation.

According to one aspect of some embodiments of the present inventionthere is provided a pharmaceutical formulation in the form of inhalabledry powder, comprising, as active ingredient a human DNase I polypeptideand particles of a physiologically acceptable pharmacologically-inertsolid carrier.

As used herein the term “dry powder” refers to a fine particulatecomposition, with particles of mean mass diameter selected capable ofbeing borne by a stream of air or gas, the dry powder not beingsuspended or dissolved in a propellant, carrier or other liquid. “Drypowder” does not necessarily imply the complete absence of watermolecules from the formulation.

As used herein, the term “physiologically inert . . . carrier” refers toa carrier whose administration to the subject does not result in aphysiological response or reaction associated with the response orreaction of the subject to the active ingredient, in this case, theDNase I. In some embodiments, the “physiologically inert . . . carrier”is devoid of any effect on the pharmaceutical activity of the activeingredient, e.g. the DNase I.

In the field of dry powders for inhalation it is customary to formulatepharmaceutically active substances with carrier particles of inertmaterial such as lactose. The carrier particles are designed such thatthey have a larger mean median diameter (MMD) than the active substanceparticles making them easier to handle and store. The smaller activeagent particles are bound to the surface of carrier particles duringstorage, but are torn from the carrier particles upon actuation of thedevice. This process is often referred to as de-agglomeration. In orderto assist in the de-agglomeration is has been proposed to employso-called force-controlling agents or anti-adherent additives inadmixture with active and carrier particles. Force controlling agentscan be surface active materials. One such force-controlling agent ismagnesium stearate. Dry powder formulations employing magnesium stearateand the like are described in U.S. Pat. No. 6,645,466, which is herebyincorporated by reference, and devices containing powders disclosed inthis patent represent particularly preferred embodiments of the presentinvention. Other force-controlling agents or anti-adherent additives aredescribed in U.S. Pat. No. 6,521,260, which is herein incorporated byreference.

In some embodiments, the inhalable dry powder formulation comprises acarrier comprising a crystalline sugar selected from the groupconsisting of glucose, arabinose, maltose, saccharose, dextrose andlactose; and at least one polyalcohol selected from the group consistingof mannitol, maltitol, lactitol and sorbitol. In one embodiment, thecrystalline sugar is lactose.

In some embodiments, the inhalable dry powder formulation comprises acarrier in the form of finely divided particles, coarse particles or acombination of fine and coarse particles. As used herein, the term“finely divided particles” includes particles of generally 0.5 to 100microns in diameter, in the range of 1-50 microns mass median diameter(MMD), in the range of 1-25 microns mass median diameter (MMD), in therange of 1-6 microns mass median diameter (MMD), 0.1-0.5 microns massmedian diameter (MMD), 0.5-1.0 microns mass median diameter (MMD),1.0-2.0 microns mass median diameter (MMD), 2.0-5 microns mass mediandiameter (MMD). As used herein, the term “coarse particles” includesparticles of generally greater than 50 microns in diameter, in the rangeof 50-500 microns mass median diameter (MMD), in the range of 150-400microns MMD. Various embodiments will entail more specific size ranges.The finely divided particles can be assemblages of crystalline plates,with the active agents entrapped or coated onto the crystalline surfacesof the particle. The particles can also be spherical shells or collapsedspherical shells with the active agent dispersed throughout. Suchparticles can be obtained by spray drying a co-solution of aco-composition (such as diketopiperazine, as described hereinbelow) andthe active agent. Other forms of particles are contemplated andencompassed by the term.

The dry powder formulation may comprise an adsorption enhancer.

The enhancer used can be any of a number of compounds which act toenhance absorption through the layer of epithelial cells lining thealveoli of the lung, and into the adjacent pulmonary vasculature. Theenhancer can accomplish this by any of several possible mechanisms:

(1) Enhancement of the paracellular permeability of DNase by inducingstructural changes in the tight junctions between the epithelial cells;

(2) Enhancement of the transcellular permeability of DNase byinteracting with or extracting protein or lipid constituents of themembrane, and thereby perturbing the membrane's integrity;

(3) Interaction between enhancer and DNase which increases thesolubility of DNase in aqueous solution. This may occur by preventingformation of DNase aggregates, or by solubilizing DNase molecules inenhancer micelies;

(4) Decreasing the viscosity of, or dissolving, the mucus barrier liningthe alveoli and passages of the lung, thereby exposing the epithelialsurface for direct absorption of the DNase.

Enhancers may function by only a single mechanism set forth above, or bytwo or more. An enhancer which acts by several mechanisms is more likelyto promote efficient absorption of DNase than one which employs only oneor two. For example, surfactants are a class of enhancers which arebelieved to act by all four mechanisms listed above. Surfactants areamphiphilic molecules having both a lipophilic and a hydrophilic moiety,with varying balance between these two characteristics. If the moleculeis very lipophilic, the low solubility of the substance in water maylimit its usefulness. If the hydrophilic part overwhelmingly dominates,however, the surface active properties of the molecule may be minimal Tobe effective, therefore, the surfactant must strike an appropriatebalance between sufficient solubility and sufficient surface activity.

In some embodiments of the invention the dry powder formulation thehuman DNase I is associated with a diketopiperazine, also acting as anadsorption enhancer. Diketopiperazines, in addition to makingaerodynamically suitable finely divided particles, also facilitatetransport across cell layers, further speeding absorption into thecirculation. Diketopiperazines can be formed into particles thatincorporate a drug or particles onto which a drug can be adsorbed. Thecombination of a drug and a diketopiperazine can impart improved drugstability. These particles can be administered by various routes ofadministration. As dry powders these particles can be delivered byinhalation to specific areas of the respiratory system, depending onparticle size. Additionally, the particles can be made small enough forincorporation into an intravenous suspension dosage form. Oral deliveryis also possible with the particles incorporated into a suspension,tablets or capsules. Diketopiperazines may also facilitate absorption ofthe DNase I.

In some embodiments of the present invention, the diketopiperazine is aderivative of 3,6-di(4-aminobutyl)-2,5-diketopiperazine, which can beformed by (thermal) condensation of the amino acid lysine. Exemplaryderivatives include 3,6-di(succinyl-4-aminobutyl)-,3,6-di(maleyl-4-aminobutyl)-, 3,6-di(glutaryl-4-aminobutyl)-,3,6-di(malonyl-4-aminobutyl)-, 3,6-di(oxalyl-4-aminobutyl)-, and3,6-di(fumaryl-4 aminobutyl)-2,5-diketopiperazine. The use ofdiketopiperazines for drug delivery is known in the art (see for exampleU.S. Pat. Nos. 5,352,461, 5,503,852, 6,071,497, and 6,331,318”, each ofwhich is incorporated herein by reference for all that it teachesregarding diketopiperazines and diketopiperazine mediated drugdelivery). Pulmonary drug delivery using finely divided diketopiperazineparticles is disclosed in U.S. Pat. No. 6,428,771, which is herebyincorporated by reference in its entirety.

As used herein the term “human DNase I protein” refers to a human DNaseI (deoxyribonuclease I; EC 3.1.21.1; DNase I) polypeptide. Human DNase Iis classified as an endonuclease, which cleaves DNA to produce 5′phosphodi- and 5′ phosphooligonucleotide end products, with a preferencefor double stranded DNA substrates and alkaline pH optimum. Othermembers of the DNase I family of endonucleases are DNase X, DNaselambda, DNASIL2 and tear lipocalin in humans.

DNase I is also known, inter alia, as alkaline DNase, bovine pancreatic(bp) DNase, DNase A, DNA phosphatase and DNA endonuclease, for example,in Bos taurus.

According to some embodiments of the invention, the human DNase Iprotein is the mature human DNase I protein, having the amino acidsequence as set forth in SEQ ID NO: 6. It will be appreciated that thehuman DNase I protein can be a modified human DNase I protein, having anamino acid sequence different than that of SEQ ID NO: 6 whilemaintaining characteristic structure and/or function of DNase I. Onenon-limiting example of a modified human DNase I protein encoded by theexpression vector of the invention is SEQ ID NO: 5.

According to yet other embodiments of the invention, the inhalable drypowder comprises a variant human DNase protein. Variant human DNaseproteins, having altered catalytic and/or other biochemical andstructural properties, such as altered actin affinity, cofactorrequirements, pH optimum, increased shelf life in storage and the like,enhanced recombinant expression or fusion proteins have been disclosed(see, for example, EC 3.1.21.2; EC 3.1.21.3; EC 3.1.21.4; EC 3.1.21.5;EC 3.1.21.6 and EC 3.1.21.7). Suitable modified DNase I polypeptidesinclude, but are not limited to DNase polypeptides disclosed in U.S.Pat. No. 6,348,343; U.S. Pat. No. 6,391,607; U.S. Pat. No. 7,407,785;U.S. Pat. No. 7,297,526 and WO2008/039989.

According to some embodiments, the inhalable dry powder formulationcomprises a DNase-like protein polypeptide. The term “DNase I-likeprotein” refers to an enzyme having an enzyme classification of EC3.1.21.x, where “x” is a positive integer. As used herein, “DNase I-likeproteins” are a subset of “DNases.” The amino acid sequences of manyDNases, and the coding sequences encoding such DNase proteins are wellknown in the art, available from public genomic databases such asGenBank, SwissProt, EMBL and many others.

According to yet further embodiments of the invention, the inhalable drypowder formulation comprises a human DNase I protein comprising anN-terminal glycine residue, for example, SEQ ID NO: 5.

According to some embodiments of the invention, the inhalable dry powderformulation comprises a recombinant human DNase I protein. As usedherein, the term “recombinant human DNase I protein” refers to a humanDNase I protein exogenously produced in a cell transformed with, andexpressing an exogenous human DNase I coding sequence. Non-limitingexamples of recombinant human DNase I protein are detailed herein,include the mammalian cell-expressed Pulmozyme® and a plant-expressedhuman recombinant DNase I (SEQ ID NO: 5).

According to some embodiments of the invention, the inhalable dry powderformulation comprises a human DNase I protein comprising the amino acidsequence as set forth in SEQ ID NO: 5, expressed in plant cells. Whenplant human recombinant DNase I (phr DNase I) was subjected tocontrolled proteolytic digestion, mass spectrometry of the resultingoligopeptides revealed that the prh DNase I polypeptide could becharacterized by a group of overlapping peptide fragments, whichtogether indicated that the full length recombinant human DNase Iexpressed by the plant cells was identical, in amino acid sequence, tothat of native human DNase I, with the addition of a N-terminal glycineresidue (see Example 2 and Tables V and Va, hereinbelow). Furtheranalysis of the amino acid sequence confirmed the accuracy of thesequence and identity with the native human DNase I. Thus, according tosome embodiments of some aspects of the invention, the inhalable drypowder formulation comprises a prh DNase I comprising a plurality ofoverlapping DNase I protein peptide fragments, the fragments having theamino acid sequence as set forth in SEQ ID NOs. 17-276 and 278-291. Insome further embodiments, the prh DNase I has an additional N-terminalglycine residue, and in other embodiments, the prh DNase I is devoid ofan N-terminal glycine residue.

According to some embodiments of some aspects of the invention, theinhalable dry powder formulation comprises a prh DNase I having amolecular mass of about 30 kD, as measured by SDS-PAGE, and about 32 kD,as measured by mass spectrometry.

In yet further embodiments, the inhalable dry powder formulationcomprises a glycosylated prh DNase polypeptide, comprising a polypeptidemoiety having a molecular mass of about 29 kD.

As shown in Examples 2 and 3, prh DNase I is fully cross-reactive withanti-human recombinant DNase I anti-serum raised against mammalian-cellexpressed recombinant human DNase I (e.g. Pulmozyme®), for example, asdetected by Western blotting of the gel-separated plant-expressedrecombinant human DNase I (see FIG. 4) and with ELISA (see Example 3).Thus, according to some embodiments, the inhalable dry powderformulation comprises a prh DNase I protein which is immunoreactive withanti-human DNase I anti-serum.

In some embodiments the prh DNase I protein is characterized by twomajor isoforms with isoelectric points between 4.2 and 4.5. In otherembodiments, the plant-expressed recombinant human DNase I protein isfurther characterized by a minor isoform with a pI between 4.2 and 3.5.

According to some embodiments of some aspects of the invention, theinhalable dry powder formulation comprises a purified prh DNase Iprotein, characterized by a purity of at least 85%, at least 87%, atleast 90%, at least 91%, at least 91.5%, at least 92%, at least 92.5%,at least 93%, at least 93.1%, at least 93.2%, at least 93.3%, at least93.4%, at least 93.5%, at least 93.6%, at least 93.7%, at least 93.8%,at least 93.9%, at least 94%, at least 94.5%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%,at least 99.7%, at least 99.8%, at least 99.9%, in a range of at least92.0-99.8% or 100% purity. In some embodiments, purity of theplant-expressed recombinant human DNase I protein is measured by HPLC.

In some embodiments the plant-expressed recombinant human DNase Icomposition comprises impurities derived from the plant host cell, suchas, but not limited to nucleic acids and polynucleotides, amino acids,oligopeptides and polypeptides, glycans and other carbohydrates, lipidsand the like. In some embodiments the host-cell derived impuritiescomprise biologically active molecules, such as enzymes.

In other embodiments, the plant-expressed recombinant human DNase Icomposition comprises plant beta-N-acetylhexosaminidase. Where the hostcell is a tobacco cell, or tobacco cell line cell, the plantbeta-N-acetylhexosaminidase is a tobacco beta-N-acetylhexosaminidase.

In further embodiments the plant beta-N-acetylhexosaminidase isinactivated plant beta-N-acetylhexosaminidase. Inactivation of plantbeta-N-acetylhexosaminidase can be effected by physical means, chemicalmeans or biochemical means. Physical inactivation can be performed byheating, freezing, desiccation, etc. Chemical inactivation can beperformed by extremes of pH, chemical denaturation, addition or removalof side chains, glycans, amino acids, etc. Biochemical inactivationincludes, but is not limited to inhibition by reversible or irreversibleinhibitors. Exemplary beta-N-acetylhexosaminidase inhibitors includeend-product inhibitors such as N-acetyl-D-glucosamine andbeta-methyl-N-acetyl glucosamine, and selective inhibitors such as thecompounds disclosed in US Patent Applications US2010016386,US20110237631, US20100087477 and US20120046337. It will be appreciatedthat preferred methods for inhibition and/or inactivation of the plantbeta-N-acetylhexosaminidase are those which also effectively preservethe structural and functional integrity of the plant-expressed humanDNase I enzyme.

In some embodiments the plant beta-N-acetylhexosaminidase is inactivatedby heating the plant-expressed recombinant human DNase I composition.Suitable temperatures for plant beta-N-acetylhexosaminidase inhibitionand/or activation include heating within a range of 37-60° C. for aperiod of 2 to 5, 10, 20, 30, 40, 50, 60 or more minutes. It will beappreciated that effective inhibition and/or inactivation of the plantbeta-N-acetylhexosaminidase is achieved more rapidly at highertemperatures and more slowly at lower temperatures of the range. IN someembodiments, the plant-expressed recombinant human DNase I compositionis heated in the range of 45-55° C. for 2-10 minutes. In someembodiments, the inhibition/inactivation results in 20, 30, 40, 50, 60,70, 80% or greater inactivation of the plantbeta-N-acetylhexosaminidase.

The present inventors have constructed a plant expression vector forrecombinant expression of human DNase I in plant cells comprising apolynucleotide encoding a human DNase I polypeptide, transformed tobaccoplants with the vector, and have isolated catalytically active humanDNase I from the plants. The biochemical properties of theplant-expressed recombinant human DNase I compare favorably with thoseof the commercially available clinical standard Pulmozyme® (Dornasealpha), and results with CF sputum suggest an advantageous effect of theplant-expressed recombinant human DNase I on the reduction ofrheological parameters of sputum, as well as reduced susceptibility toactin inhibition of the DNase endonuclease activity. Structuraldifferences uncovered between the plant-expressed recombinant humanDNase I and mammalian-cell expressed recombinant human DNase I (e.g.Pulmozyme®) (mobility on PAGE, unity of immunoreactive DNase I speciesas detected on Western blots, see hereinbelow) suggest that bothmodifications of amino acid sequence and post translational modificationresulting from processing of the expressed polypeptide by the plant cellmay be responsible for some of the distinguishing functionalcharacteristics of the plant-expressed recombinant enzyme.

Thus, in some embodiments, there is provided an isolated polynucleotidecomprising a nucleic acid sequence encoding a human DNase I protein,wherein the human DNase I protein is contiguously linked at theN-terminal to a plant endoplasmic reticulum targeting signal peptide.

According to another aspect of the invention, the human DNase I proteinis contiguously linked at the N-terminal to an Arabidopsis ABPIendoplasmic reticulum targeting signal peptide.

As used herein the term “contiguously linked at the N-terminal” refersto covalent attachment of the indicated peptide via a peptide bond tothe N-terminal amino acid of the mature protein, for example, humanDNase I protein. “Contiguously linked at the C-terminal” refers tocovalent attachment of the indicated peptide via a peptide bond to theC-terminal amino acid of the mature protein, for example, human DNase Iprotein.

As used herein, the term “Arabidopsis ABPI endoplasmic reticulumtargeting signal peptide” refers to the leader peptide sequence of theArabidopsis thaliana auxin binding protein, which is capable ofdirecting the expressed protein to the endoplasmic reticulum within theplant cell. In one embodiment, the Arabidopsis ABPI endoplasmicreticulum targeting signal peptide is a 33 amino acid polypeptide as setforth in SEQ ID NO: 4.

Thus, according to another aspect of the present invention, the humanDNase I protein contiguously linked at the N-terminal to an ArabidopsisABPI endoplasmic reticulum targeting signal peptide and the human DNaseI protein has an amino acid sequence as set forth in SEQ ID NO: 1.

According to some embodiments of the invention, the human DNase Iprotein is encoded by a nucleic acid sequence as set forth in SEQ ID NO:9. According to further embodiments of the invention, the ArabidopsisABPI endoplasmic reticulum targeting signal peptide is encoded by anucleic acid sequence as set forth in SEQ ID NO: 10.

According to still further embodiments of the invention the human DNaseI protein contiguously linked at the N-terminal to an Arabidopsis ABPIendoplasmic reticulum targeting signal peptide is encoded by a nucleicacid sequence as set forth in SEQ ID NO: 12.

In order to express the polypeptide, the sequence encoding same isligated into a “plant nucleic acid expression construct”.

As used herein the term “plant nucleic acid expression construct” refersto a nucleic acid construct which includes the nucleic acid of someembodiments of the invention and at least one promoter for directingtranscription of nucleic acid in a host plant cell. Further details ofsuitable transformation approaches are provided hereinbelow.

According to some embodiments of the invention, there is provided anucleic acid expression construct comprising the nucleic acid sequenceof the invention, and a promoter for directing transcription of thenucleic acid sequence in a plant host cell.

As used herein the term “nucleic acid sequence” refers to a single ordouble stranded nucleic acid sequence which is isolated and provided inthe form of an RNA sequence, a complementary polynucleotide sequence(cDNA), a genomic polynucleotide sequence and/or a compositepolynucleotide sequences (e.g., a combination of the above).

As used herein the phrase “complementary polynucleotide sequence” refersto a sequence, which results from reverse transcription of messenger RNAusing a reverse transcriptase or any other RNA dependent DNA polymerase.Such a sequence can be subsequently amplified in vivo or in vitro usinga DNA dependent DNA polymerase.

As used herein the phrase “genomic polynucleotide sequence” refers to asequence derived (isolated) from a chromosome and thus it represents acontiguous portion of a chromosome.

As used herein the phrase “composite polynucleotide sequence” refers toa sequence, which is at least partially complementary and at leastpartially genomic. A composite sequence can include some exonalsequences required to encode the polypeptide of the present invention,as well as some intronic sequences interposing therebetween. Theintronic sequences can be of any source, including of other genes, andtypically will include conserved splicing signal sequences. Suchintronic sequences may further include cis acting expression regulatoryelements.

According to some embodiments of the present invention, the nucleic acidsequences encoding the polypeptides of the present invention areoptimized for expression in plants. Examples of such sequencemodifications include, but are not limited to, an altered G/C content tomore closely approach that typically found in the plant species ofinterest, and the removal of codons atypically found in the plantspecies commonly referred to as codon optimization. In one embodiment,the codon usage of the nucleic acid sequence encoding the human DNase Iprotein, the human DNase I protein contiguously linked at the N-terminalto an Arabidopsis ABPI endoplasmic reticulum targeting signal peptide,or any other human DNase I protein described herein is optimized forNicotiana tabacuum or Nicotiana benthamiana.

The phrase “codon optimization” refers to the selection of appropriateDNA nucleotides for use within a structural gene or fragment thereofthat approaches codon usage within the plant of interest. Therefore, anoptimized gene or nucleic acid sequence refers to a gene in which thenucleotide sequence of a native or naturally occurring gene has beenmodified in order to utilize statistically-preferred orstatistically-favored codons within the plant. The nucleotide sequencetypically is examined at the DNA level and the coding region optimizedfor expression in the plant species determined using any suitableprocedure, for example as described in Sardana et al. (1996, Plant CellReports 15:677-681). In this method, the standard deviation of codonusage, a measure of codon usage bias, may be calculated by first findingthe squared proportional deviation of usage of each codon of the nativegene relative to that of highly expressed plant genes, followed by acalculation of the average squared deviation. The formula used is: 1SDCU=n=1N[(Xn−Yn)/Yn]2/N, where Xn refers to the frequency of usage ofcodon n in highly expressed plant genes, where Yn to the frequency ofusage of codon n in the gene of interest and N refers to the totalnumber of codons in the gene of interest. A table of codon usage fromhighly expressed genes of dicotyledonous plants has been compiled usingthe data of Murray et al. (1989, Nuc Acids Res. 17:477-498).

One method of optimizing the nucleic acid sequence in accordance withthe preferred codon usage for a particular plant cell type is based onthe direct use, without performing any extra statistical calculations,of codon optimization tables such as those provided on-line at the CodonUsage Database through the NIAS (National Institute of AgrobiologicalSciences) DNA bank in Japan (Hypertext Transfer Protocol://World WideWeb (dot) kazusa (dot) or (dot) jp/codon/). The Codon Usage Databasecontains codon usage tables for a number of different species, with eachcodon usage table having been statistically determined based on the datapresent in Genbank.

By using such codon optimization tables to determine the most preferredor most favored codons for each amino acid in a particular species (forexample, rice), a naturally-occurring nucleotide sequence encoding aprotein of interest can be codon optimized for that particular plantspecies. This is effected by replacing codons that may have a lowstatistical incidence in the particular species genome withcorresponding codons, in regard to an amino acid, that are statisticallymore favored. However, one or more less-favored codons may be selectedto delete existing restriction sites, to create new ones at potentiallyuseful junctions (5′ and 3′ ends to add signal peptide or terminationcassettes, internal sites that might be used to cut and splice segmentstogether to produce a correct full-length sequence), or to eliminatenucleotide sequences that may negatively effect mRNA stability orexpression.

The desired encoding nucleotide sequence may already, in advance of anymodification, contain a number of codons that correspond to astatistically-favored codon in a particular plant species. Therefore,codon optimization of the native nucleotide sequence may comprisedetermining which codons, within the desired nucleotide sequence, arenot statistically-favored with regards to a particular plant, andmodifying these codons in accordance with a codon usage table of theparticular plant to produce a codon optimized derivative. A modifiednucleotide sequence may be fully or partially optimized for plant codonusage provided that the protein encoded by the modified nucleotidesequence is produced at a level higher than the protein encoded by thecorresponding naturally occurring or native gene. Construction ofsynthetic genes by altering the codon usage is described in for examplePCT Patent Application 93/07278.

According to some embodiments of the invention, the nucleic acid isoperably linked to the promoter sequence.

A coding nucleic acid sequence is “operably linked” to a regulatorysequence (e.g., promoter) if the regulatory sequence is capable ofexerting a regulatory effect on (e.g. effect on the expression of) thecoding sequence linked thereto.

Any suitable promoter sequence can be used by the nucleic acid constructof the present invention. Preferably the promoter is a constitutivepromoter, a tissue-specific, or an inducible promoter.

As used herein the phrase “plant-expressible” refers to a promotersequence, including any additional regulatory elements added thereto orcontained therein, is at least capable of inducing, conferring,activating or enhancing expression in a plant cell, tissue or organ,preferably a monocotyledonous or dicotyledonous plant cell, tissue, ororgan. Such a promoter can be constitutive, i.e., capable of directinghigh level of gene expression in a plurality of tissues, tissuespecific, i.e., capable of directing gene expression in a particulartissue or tissues, inducible, i.e., capable of directing gene expressionunder a stimulus, or chimeric, i.e., formed of portions of at least twodifferent promoters.

Examples of preferred promoters useful for the methods of someembodiments of the invention are presented in Table I, II, III and IV.

TABLE I Exemplary constitutive promoters for use in the performance ofsome embodiments of the invention Gene Source Expression PatternReference Actin constitutive McElroy etal, Plant Cell, 2: 163-171, 1990CAMV 35S constitutive Odell et al, Nature, 313: 810-812, 1985 CaMV 19Sconstitutive Nilsson et al., Physiol. Plant 100: 456-462, 1997 GOS2constitutive de Pater et al, Plant J Nov; 2(6): 837-44, 1992 ubiquitinconstitutive Christensen et al, Plant Mol. Biol. 18: 675-689, 1992 Ricecyclophilin constitutive Bucholz et al, Plant Mol Biol. 25(5): 837-43,1994 Maize H3 histone constitutive Lepetit et al, Mol. Gen. Genet. 231:276-285, 1992 Actin 2 constitutive An et al, Plant J. 10(1); 107- 121,1996

TABLE II Exemplary seed-preferred promoters for use in the performanceof some embodiments of the invention Gene Source Expression PatternReference Seed specific genes seed Simon, et al., Plant Mol. Biol. 5.191, 1985; Scofield, etal., J. Biol. Chem. 262: 12202, 1987.;Baszczynski, et al., Plant Mol. Biol. 14: 633, 1990. Brazil Nut albuminseed Pearson′ et al., Plant Mol. Biol. 18: 235-245, 1992. legumin seedEllis, et al. Plant Mol. Biol. 10: 203-214, 1988 Glutelin (rice) seedTakaiwa, et al., Mol. Gen. Genet. 208: 15-22, 1986; Takaiwa, et al.,FEBS Letts. 221: 43-47, 1987 Zein seed Matzke et al Plant Mol Biol,143). 323-32 1990 napA seed Stalberg, et al, Planta 199: 515-519, 1996wheat LMW and endosperm Mol Gen Genet 216: HMW, glutenin-1 81-90, 1989;NAR 17: 461-2, Wheat SPA seed Albanietal, Plant Cell, 9: 171-184, 1997wheat a, b and endosperm EMBO3: 1409-15, 1984 g gliadins Barley ltrlpromoter endosperm barley B1, C, endosperm Theor Appl Gen 98: 1253- Dhordein 62, 1999; Plant J 4: 343- 55, 1993; Mol Gen Genet 250: 750-60,1996 Barley DOF endosperm Mena et al, The Plant Journal, 116(1): 53-62,1998 Biz2 endosperm EP99106056.7 Synthetic promoter endospermVicente-Carbajosa et al., Plant J. 13: 629-640, 1998 rice prolaminendosperm Wu et al, Plant Cell NRP33 Physiology 39(8) 885-889, 1998 rice-globulin endosperm Wu et al, Plant Cell Glb-1 Physiology 398) 885-889,1998 rice OSH1 embryo Sato et al, Proc. Nati. Acad. Sci. USA, 93:8117-8122 rice alpha-globulin endosperm Nakase et al. Plant Mol.REB/OHP-1 Biol. 33: 513-S22, 1997 rice ADP- endosperm Trans Res 6:157-68, 1997 glucose PP maize ESR endosperm Plant J 12: 235-46, 1997gene family sorghum gamma- endosperm PMB 32: 1029-35, 1996 kafirin KNOXemryo Postma-Haarsma ef al, Plant Mol. Biol. 39: 257- 71, 1999 riceoleosin Embryo and aleuton Wu et at, J. Biochem., 123: 386, 1998sunflower Seed (embryo and dry Cummins, etal., Plant Mol. oleosin seed)Biol. 19: 873-876, 1992

TABLE III Exemplary flower-specific promoters for use in the performanceof the invention Expression Gene Source Pattern Reference AtPRP4 flowerswww.salus. medium.edu/m mg/tierney/html chalene synthase (chsA) flowersVan der Meer, et al., Plant Mol. Biol. 15, 95-109, 1990. LAT52 antherTwell et al Mol. Gen Genet. 217: 240-245 (1989) apetala- 3 flowers

TABLE IV Alternative rice promoters for use in the performance of theinvention PRO # gene expression PR00001 Metallothionein Mte transferlayer of embryo + calli PR00005 putative beta-amylase transfer layer ofembryo PR00009 Putative cellulose synthase Weak in roots PR00012 lipase(putative) PR00014 Transferase (putative) PR00016 peptidyl prolylcis-trans isomerase (putative) PR00019 unknown PR00020 prp protein(putative) PR00029 noduline (putative) PR00058 Proteinase inhibitorRgpi9 seed PR00061 beta expansine EXPB9 Weak in young flowers PR00063Structural protein young tissues + calli + embryo PR00069 xylosidase(putative) PR00075 Prolamine 10 Kda strong in endosperm PR00076 allergenRA2 strong in endosperm PR00077 prolamine RP7 strong in endospermPR00078 CBP80 PR00079 starch branching enzyme I PR00080Metallothioneine-like ML2 transfer layer of embryo + calli PR00081putative caffeoyl- CoA shoot 3-0 methyltransferase PR00087 prolamine RM9strong in endosperm PR00090 prolamine RP6 strong in endosperm PR00091prolamine RP5 strong in endosperm PR00092 allergen RA5 PR00095 putativemethionine embryo aminopeptidase PR00098 ras-related GTP binding proteinPR00104 beta expansine EXPB1 PR00105 Glycine rich protein PR00108metallothionein like protein (putative) PR00110 RCc3 strong root PR00111uclacyanin 3-like protein weak discrimination center/shoot meristemPR00116 26S proteasome regulatory very weak meristem specific particlenon-ATPase subunit 11 PR00117 putative 40S ribosomal weak in endospermprotein PR00122 chlorophyll a/lo-binding very weak in shoot proteinprecursor (Cab27) PR00123 putative protochlorophyllide Strong leavesreductase PR00126 metallothionein RiCMT strong discrimination centershoot meristem PR00129 GOS2 Strong constitutive PR00131 GOS9 PR00133chitinase Cht-3 very weak meristem specific PR00135 alpha- globulinStrong in endosperm PR00136 alanine aminotransferase Weak in endospermPR00138 Cyclin A2 PR00139 Cyclin D2 PR00140 Cyclin D3 PR00141Cyclophyllin 2 Shoot and seed PR00146 sucrose synthase SS1 (barley)medium constitutive PR00147 trypsin inhibitor ITR1 (barley) weak inendosperm PR00149 ubiquitine 2 with intron strong constitutive PR00151WSI18 Embryo and stress PR00156 HVA22 homologue (putative) PR00157 EL2PR00169 aquaporine medium constitutive in young plants PR00170 Highmobility group protein Strong constitutive PR00171 reversiblyglycosylated weak constitutive protein RGP1 PR00173 cytosolic MDH shootPR00175 RAB21 Embryo and stress PR00176 CDPK7 PR00177 Cdc2-l very weakin meristem PR00197 sucrose synthase 3 PRO0198 OsVP1 PRO0200 OSH1 veryweak in young plant meristem PRO0208 putative chlorophyllase PRO0210OsNRT1 PRO0211 EXP3 PRO0216 phosphate transporter OjPT1 PRO0218 oleosin18 kd aleurone + embryo PRO0219 ubiquitine 2 without intron PRO0220 RFLPRO0221 maize UBI delta intron not detected PRO0223 glutelin-1 PRO0224fragment of prolamin RP6 promoter PRO0225 4xABRE PRO0226 glutelinOSGLUA3 PRO0227 BLZ-2_short (barley) PR00228 BLZ-2_long (barley)

The nucleic acid construct of some embodiments of the invention canfurther include an appropriate selectable marker and/or an origin ofreplication. According to some embodiments of the invention, the nucleicacid construct utilized is a shuttle vector, which can propagate both inE. coli (wherein the construct comprises an appropriate selectablemarker and origin of replication) and be compatible with propagation incells. The construct according to the present invention can be, forexample, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus oran artificial chromosome.

The nucleic acid construct of some embodiments of the invention can beutilized to stably or transiently transform plant cells. In stabletransformation, the nucleic acid is integrated into the plant genome andas such it represents a stable and inherited trait. In transienttransformation, the exogenous polynucleotide is expressed by the celltransformed but it is not integrated into the genome and as such itrepresents a transient trait.

Thus, according to some aspects of the present invention, there isprovided an isolated cell comprising the nucleic acid construct of theinvention.

As used herein, the term “isolated cell” refers to a cell at leastpartially separated from the natural environment e.g., from a plant. Insome embodiments, the isolated cell is a plant cell of a whole plant. Insome embodiments, the isolated cell is a plant cell, for example, aplant cell in culture.

The term “plant” as used herein encompasses whole plants, ancestors andprogeny of the plants and plant parts, including seeds, shoots, stems,roots (including tubers), and plant cells, tissues and organs. The plantmay be in any form including suspension cultures, embryos, meristematicregions, callus tissue, leaves, gametophytes, sporophytes, pollen, andmicrospores. Plants that are particularly useful in the methods of theinvention include all plants which belong to the superfamilyViridiplantae, in particular monocotyledonous and dicotyledonous plantsincluding a fodder or forage legume, ornamental plant, food crop, tree,or shrub selected from the list comprising Acacia spp., Acer spp.,Actinidia spp., Aesculus spp., Agathis australis, Albizia amara,Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Asteliafragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassicaspp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadabafarinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicumspp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomumcassia, Coffea arabica, Colophospermum mopane, Coronillia varia,Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp.,Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogonspp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davalliadivaricata, Desmodium spp., Dicksonia squarosa, Dibeteropogonamplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloapyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp.,Erythrina spp., Eucalypfus spp., Euclea schimperi, Eulalia vi/losa,Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp,Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycinejavanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtiacoleosperma, Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus,Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffheliadissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia,Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex,Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihotesculenta, Medicago saliva, Metasequoia glyptostroboides, Musasapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryzaspp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petuniaspp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photiniaspp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara,Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopiscineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis,Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhusnatalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosaspp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitysvefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghumbicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides,Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themedatriandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vacciniumspp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschiaaethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brusselssprouts, cabbage, canola, carrot, cauliflower, celery, collard greens,flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean,straw, sugar beet, sugar cane, sunflower, tomato, squash tea, maize,wheat, barely, rye, oat, peanut, pea, lentil and alfalfa, cotton,rapeseed, canola, pepper, sunflower, tobacco, eggplant, eucalyptus, atree, an ornamental plant, a perennial grass and a forage crop.Alternatively algae and other non-Viridiplantae can be used for themethods of the present invention.

According to some embodiments of the invention, the plant or plant cellis a duckweed plant, cell or nodule. Duckweed (members of themonocotyledonous family Lemnaceae, or Lemna) plant or duckweed nodulecultures can be efficiently transformed with an expression cassettecontaining a nucleotide sequence of interest by any one of a number ofmethods including Agrobacterium-mediated gene transfer, ballisticbombardment, or electroporation. Methods for molecular engineering ofduckweed cells and detailed description of duckweed expression systemsand useful for commercial production of valuable polypeptides are knownin the art (see, for example, U.S. Pat. Nos. 6,040,498 and 6,815,184 toStomp, et al, and U.S. Pat. No. 8,022,270 to Dickey et al).

According to some embodiments of the invention, the plant or plant cellused by the method of the invention is a crop plant or cell of a cropplant such as rice, maize, wheat, barley, peanut, potato, sesame, olivetree, palm oil, banana, soybean, sunflower, canola, sugarcane, alfalfa,millet, leguminosae (bean, pea), flax, lupinus, rapeseed, tobacco,poplar and cotton.

According to further embodiments the plant cells includes tobacco cells,Agrobacterium rihzogenes transformed root cell, celery cell, gingercell, horseradish cell and carrot cells. In one embodiment the tobaccocells are from a tobacco cell line, such as, but not limited toNicotiana tabacum L. cv Bright Yellow (BY-2) cells. The plant cells maybe grown according to any type of suitable culturing method, includingbut not limited to, culture on a solid surface (such as a plasticculturing vessel or plate for example) or in suspension. It will benoted that some cells, such as the BY-2 and carrot cells can be culturedand grown in suspension. Suitable devices and methods for culturingplant cells in suspension are known in the art, for example, asdescribed in International Patent Application PCT IL2008/000614. In yetanother embodiment the cells are cells of whole tobacco plants or planttissues, including, but not limited to Nicotiana benthamiana.

There are various methods of introducing foreign genes into bothmonocotyledonous and dicotyledonous plants (Potrykus, I., Annu. Rev.Plant. Physiol., Plant. Mol. Biol. (1991) 42:205-225; Shimamoto et al.,Nature (1989) 338:274-276).

The principle methods of causing stable integration of exogenous DNAinto plant genomic DNA include two main approaches:

-   -   (i) Agrobacterium-mediated gene transfer: Klee et al. (1987)        Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogers in Cell        Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular        Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L.        K., Academic Publishers, San Diego, Calif. (1989) p. 2-25;        Gatenby, in Plant Biotechnology, eds. Kung, S. and Amtzen, C.        J., Butterworth Publishers, Boston, Mass. (1989) p. 93-112.    -   (ii) Direct DNA uptake: Paszkowski et al., in Cell Culture and        Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of        Plant Nuclear Genes eds. Schell, J., and Vasil, L. K., Academic        Publishers, San Diego, Calif. (1989) p. 52-68; including methods        for direct uptake of DNA into protoplasts, Toriyama, K. et        al. (1988) Bio/Technology 6:1072-1074. DNA uptake induced by        brief electric shock of plant cells: Zhang et al. Plant Cell        Rep. (1988) 7:379-384. Fromm et al. Nature (1986) 319:791-793.        DNA injection into plant cells or tissues by particle        bombardment, Klein et al. Bio/Technology (1988) 6:559-563;        McCabe et al. Bio/Technology (1988) 6:923-926; Sanford, Physiol.        Plant. (1990) 79:206-209; by the use of micropipette systems:        Neuhaus et al., Theor. Appl. Genet. (1987) 75:30-36; Neuhaus and        Spangenberg, Physiol. Plant. (1990) 79:213-217; glass fibers or        silicon carbide whisker transformation of cell cultures, embryos        or callus tissue, U.S. Pat. No. 5,464,765 or by the direct        incubation of DNA with germinating pollen, DeWet et al. in        Experimental Manipulation of Ovule Tissue, eds. Chapman, G. P.        and Mantell, S. H. and Daniels, W. Longman, London, (1985) p.        197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.

The Agrobacterium system includes the use of plasmid vectors thatcontain defined DNA segments that integrate into the plant genomic DNA.Methods of inoculation of the plant tissue vary depending upon the plantspecies and the Agrobacterium delivery system. A widely used approach isthe leaf disc procedure which can be performed with any tissue explantthat provides a good source for initiation of whole plantdifferentiation. See, e.g., Horsch et al. in Plant Molecular BiologyManual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. Asupplementary approach employs the Agrobacterium delivery system incombination with vacuum infiltration. The Agrobacterium system isespecially viable in the creation of transgenic dicotyledonous plants.

There are various methods of direct DNA transfer into plant cells. Inelectroporation, the protoplasts are briefly exposed to a strongelectric field. In microinjection, the DNA is mechanically injecteddirectly into the cells using very small micropipettes. In microparticlebombardment, the DNA is adsorbed on microprojectiles such as magnesiumsulfate crystals or tungsten particles, and the microprojectiles arephysically accelerated into cells or plant tissues.

Following stable transformation plant propagation is exercised. The mostcommon method of plant propagation is by seed. Regeneration by seedpropagation, however, has the deficiency that due to heterozygositythere is a lack of uniformity in the crop, since seeds are produced byplants according to the genetic variances governed by Mendelian rules.Basically, each seed is genetically different and each will grow withits own specific traits. Therefore, it is preferred that the transformedplant be produced such that the regenerated plant has the identicaltraits and characteristics of the parent transgenic plant. Therefore, itis preferred that the transformed plant be regenerated bymicropropagation which provides a rapid, consistent reproduction of thetransformed plants.

Micropropagation is a process of growing new generation plants from asingle piece of tissue that has been excised from a selected parentplant or cultivar. This process permits the mass reproduction of plantshaving the preferred tissue expressing the fusion protein. The newgeneration plants which are produced are genetically identical to, andhave all of the characteristics of, the original plant. Micropropagationallows mass production of quality plant material in a short period oftime and offers a rapid multiplication of selected cultivars in thepreservation of the characteristics of the original transgenic ortransformed plant. The advantages of cloning plants are the speed ofplant multiplication and the quality and uniformity of plants produced.

Micropropagation is a multi-stage procedure that requires alteration ofculture medium or growth conditions between stages. Thus, themicropropagation process involves four basic stages: Stage one, initialtissue culturing; stage two, tissue culture multiplication; stage three,differentiation and plant formation; and stage four, greenhouseculturing and hardening. During stage one, initial tissue culturing, thetissue culture is established and certified contaminant-free. Duringstage two, the initial tissue culture is multiplied until a sufficientnumber of tissue samples are produced to meet production goals. Duringstage three, the tissue samples grown in stage two are divided and growninto individual plantlets. At stage four, the transformed plantlets aretransferred to a greenhouse for hardening where the plants' tolerance tolight is gradually increased so that it can be grown in the naturalenvironment.

According to some embodiments of the invention, the transgenic plantsare generated by transient transformation of leaf cells, meristematiccells or the whole plant.

Transient transformation can be effected by any of the direct DNAtransfer methods described above or by viral infection using modifiedplant viruses.

Viruses that have been shown to be useful for the transformation ofplant hosts include CaMV, Tobacco mosaic virus (TMV), brome mosaic virus(BMV) and Bean Common Mosaic Virus (BV or BCMV). Transformation ofplants using plant viruses is described in U.S. Pat. No. 4,855,237 (beangolden mosaic virus; BGV), EP-A 67,553 (TMV), Japanese PublishedApplication No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); andGluzman, Y. et al., Communications in Molecular Biology: Viral Vectors,Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudovirusparticles for use in expressing foreign DNA in many hosts, includingplants are described in WO 87/06261.

According to some embodiments of the invention, the virus used fortransient transformations is avirulent and thus is incapable of causingsevere symptoms such as reduced growth rate, mosaic, ring spots, leafroll, yellowing, streaking, pox formation, tumor formation and pitting.A suitable avirulent virus may be a naturally occurring avirulent virusor an artificially attenuated virus. Virus attenuation may be effectedby using methods well known in the art including, but not limited to,sub-lethal heating, chemical treatment or by directed mutagenesistechniques such as described, for example, by Kurihara and Watanabe(Molecular Plant Pathology 4:259-269, 2003), Galon et al. (1992), Atreyaet al. (1992) and Huet et al. (1994).

Suitable virus strains can be obtained from available sources such as,for example, the American Type culture Collection (ATCC) or by isolationfrom infected plants. Isolation of viruses from infected plant tissuescan be effected by techniques well known in the art such as described,for example by Foster and Tatlor, Eds. “Plant Virology Protocols: FromVirus Isolation to Transgenic Resistance (Methods in Molecular Biology(Humana Pr), Vol 81)”, Humana Press, 1998. Briefly, tissues of aninfected plant believed to contain a high concentration of a suitablevirus, preferably young leaves and flower petals, are ground in a buffersolution (e.g., phosphate buffer solution) to produce a virus infectedsap which can be used in subsequent inoculations.

Construction of plant RNA viruses for the introduction and expression ofnon-viral nucleic acid sequences in plants is demonstrated by the abovereferences as well as by Dawson, W. O. et al., Virology (1989)172:285-292; Takamatsu et al. EMBO J. (1987) 6:307-311; French et al.Science (1986) 231:1294-1297; Takamatsu et al. FEBS Letters (1990)269:73-76; and U.S. Pat. No. 5,316,931.

When the virus is a DNA virus, suitable modifications can be made to thevirus itself. Alternatively, the virus can first be cloned into abacterial plasmid for ease of constructing the desired viral vector withthe foreign DNA. The virus can then be excised from the plasmid. If thevirus is a DNA virus, a bacterial origin of replication can be attachedto the viral DNA, which is then replicated by the bacteria.Transcription and translation of this DNA will produce the coat proteinwhich will encapsidate the viral DNA. If the virus is an RNA virus, thevirus is generally cloned as a cDNA and inserted into a plasmid. Theplasmid is then used to make all of the constructions. The RNA virus isthen produced by transcribing the viral sequence of the plasmid andtranslation of the viral genes to produce the coat protein(s) whichencapsidate the viral RNA.

In one embodiment, a plant viral polynucleotide is provided in which thenative coat protein coding sequence has been deleted from a viralpolynucleotide, a non-native plant viral coat protein coding sequenceand a non-native promoter, preferably the subgenomic promoter of thenon-native coat protein coding sequence, capable of expression in theplant host, packaging of the recombinant plant viral polynucleotide, andensuring a systemic infection of the host by the recombinant plant viralpolynucleotide, has been inserted. Alternatively, the coat protein genemay be inactivated by insertion of the non-native polynucleotidesequence within it, such that a protein is produced. The recombinantplant viral polynucleotide may contain one or more additional non-nativesubgenomic promoters. Each non-native subgenomic promoter is capable oftranscribing or expressing adjacent genes or polynucleotide sequences inthe plant host and incapable of recombination with each other and withnative subgenomic promoters. Non-native (foreign) polynucleotidesequences may be inserted adjacent the native plant viral subgenomicpromoter or the native and a non-native plant viral subgenomic promotersif more than one polynucleotide sequence is included. The non-nativepolynucleotide sequences are transcribed or expressed in the host plantunder control of the subgenomic promoter to produce the desiredproducts.

In a second embodiment, a recombinant plant viral polynucleotide isprovided as in the first embodiment except that the native coat proteincoding sequence is placed adjacent one of the non-native coat proteinsubgenomic promoters instead of a non-native coat protein codingsequence.

In a third embodiment, a recombinant plant viral polynucleotide isprovided in which the native coat protein gene is adjacent itssubgenomic promoter and one or more non-native subgenomic promoters havebeen inserted into the viral polynucleotide. The inserted non-nativesubgenomic promoters are capable of transcribing or expressing adjacentgenes in a plant host and are incapable of recombination with each otherand with native subgenomic promoters. Non-native polynucleotidesequences may be inserted adjacent the non-native subgenomic plant viralpromoters such that the sequences are transcribed or expressed in thehost plant under control of the subgenomic promoters to produce thedesired product.

In a fourth embodiment, a recombinant plant viral polynucleotide isprovided as in the third embodiment except that the native coat proteincoding sequence is replaced by a non-native coat protein codingsequence.

The viral vectors are encapsidated by the coat proteins encoded by therecombinant plant viral polynucleotide to produce a recombinant plantvirus. The recombinant plant viral polynucleotide or recombinant plantvirus is used to infect appropriate host plants. The recombinant plantviral polynucleotide is capable of replication in the host, systemicspread in the host, and transcription or expression of foreign gene(s)(exogenous polynucleotide) in the host to produce the desired protein.

Techniques for inoculation of viruses to plants may be found in Fosterand Taylor, eds. “Plant Virology Protocols: From Virus Isolation toTransgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol81)”, Humana Press, 1998; Maramorosh and Koprowski, eds. “Methods inVirology” 7 vols, Academic Press, New York 1967-1984; Hill, S. A.“Methods in Plant Virology”, Blackwell, Oxford, 1984; Walkey, D. G. A.“Applied Plant Virology”, Wiley, New York, 1985; and Kado and Agrawa,eds. “Principles and Techniques in Plant Virology”, VanNostrand-Reinhold, New York.

In addition to the above, the polynucleotide of the present inventioncan also be introduced into a chloroplast genome thereby enablingchloroplast expression.

A technique for introducing exogenous nucleic acid sequences to thegenome of the chloroplasts is known. This technique involves thefollowing procedures. First, plant cells are chemically treated so as toreduce the number of chloroplasts per cell to about one. Then, theexogenous polynucleotide is introduced via particle bombardment into thecells with the aim of introducing at least one exogenous polynucleotidemolecule into the chloroplasts. The exogenous polynucleotides selectedsuch that it is integratable into the chloroplast's genome viahomologous recombination which is readily effected by enzymes inherentto the chloroplast. To this end, the nucleic acid sequence includes, inaddition to a gene of interest, at least one polynucleotide stretchwhich is derived from the chloroplast's genome. In addition, theexogenous polynucleotide includes a selectable marker, which serves bysequential selection procedures to ascertain that all or substantiallyall of the copies of the chloroplast genomes following such selectionwill include the exogenous polynucleotide. Further details relating tothis technique are found in U.S. Pat. Nos. 4,945,050; and 5,693,507which are incorporated herein by reference. A polypeptide can thus beproduced by the protein expression system of the chloroplast and becomeintegrated into the chloroplast's inner membrane.

According to some embodiments of the invention, the method furthercomprises growing the plant cell expressing the nucleic acid. The plantcells can be any plant cells desired. The plant cells can be culturedcells, cells in cultured tissue or cultured organs, or cells in a plant.In some embodiments, the plant cells are cultured cells, or cells incultured tissue or cultured organs. In yet further embodiments, theplant cells are any type of plant that is used in gene transference. Theplant cell can be grown as part of a whole plant, or, alternatively, inplant cell culture.

According to some aspects of the invention, the plant cells are grown ina plant cell suspension culture. As used herein, the term “suspensionculture” refers to the growth of cells separate from the organism.Suspension culture can be facilitated via use of a liquid medium (a“suspension medium”). Suspension culture can refer to the growth ofcells in liquid nutrient media. Methods and devices suitable for growingplant cells of the invention in plant cell suspension culture aredescribed in detail in, for example, PCT WO2008/135991, U.S. Pat. No.6,391,683, U.S. patent application Ser. No. 10/784,295; InternationalPatent Publications PCT Nos. WO2004/091475, WO2005/080544 and WO2006/040761, all of which are hereby incorporated by reference as iffully set forth herein.

Thus, the invention encompasses plants or plant cultures expressing thenucleic acid sequences, so as to produce the recombinant human DNase Iprotein of the invention. Once expressed within the plant cell or theentire plant, the level of the human DNase I protein encoded by thenucleic acid sequence can be determined by methods well known in the artsuch as, activity assays, Western blots using antibodies capable ofspecifically binding the human DNase I protein, Enzyme-Linked ImmunoSorbent Assay (ELISA), radio-immuno-assays (RIA), immunohistochemistry,immunocytochemistry, immunofluorescence and the like.

Methods of determining the level in the plant of the RNA transcribedfrom the nucleic acid sequence are well known in the art and include,for example, Northern blot analysis, reverse transcription polymerasechain reaction (RT-PCR) analysis (including quantitative,semi-quantitative or real-time RT-PCR) and RNA-in situ hybridization.

According to some embodiments of the invention, the expressedrecombinant human DNase I protein is glycosylated in the plant cell,resulting in a recombinant human DNase I protein having high mannoseglycosylation (e.g. exposed mannose sugar residues), and plant specificglycan residues. Thus, according to some embodiments of the invention,the cells expressing the expression vector of the invention produce ahuman DNase I protein having at least one, optionally at least two,optionally at least three or optionally at least four or more exposedmannose residues. In other embodiments the cells expressing theexpression vector of the invention produce a human DNase I proteinhaving at least one, optionally at least two, optionally at least threeor optionally at least four or more core xylose residues. In yet otherembodiments the cells expressing the expression vector of the inventionproduce a human DNase I protein having at least one, optionally at leasttwo, optionally at least three or optionally at least four or more coreα-(1,3) fucose residues. In one embodiment the cells expressing theexpression vector of the invention produce a human DNase I proteinhaving at least one exposed mannose residue, at least one core xyloseresidue and at least one α-(1,3) fucose residue. In yet furtherembodiments, the cells expressing the expression vector of the inventionproduce a human DNase I protein having at least one, at least two, atleast 3 or more terminal N-acetyl glucosamine substitutions on the outermannose sugars.

Glycan analysis of human DNase I protein expressed in plant cellsindicated that the prh DNase I lacks sialic acid residues. Thus, in yetother embodiments, the cells expressing the expression vector of theinvention produce a recombinant human DNase I protein devoid of sialicacid residues.

The human DNase I protein produced by cells expressing the expressionvector of the invention was shown to have DNase I catalytic activitysimilar or superior to that of a native human or mammalian-cell producedDNase I enzyme.

As shown in Example 3 herein, when enzyme kinetics of theplant-expressed recombinant human DNase I protein were measured (seeTable VI), greater substrate affinity (K_(M)) and higher reactionvelocity (V_(max)) were observed for the plant-expressed recombinanthuman DNase I protein, as compared to those of mammalian cell-expressedrecombinant human DNase I (e.g. Pulmozyme®). Thus, according to someembodiments the plant expressed recombinant human DNase I isbiologically active. In some embodiments, the biological activity isendonuclease catalytic activity. In yet other embodiments, thebiological activity of the plant-expressed recombinant human DNase Iprotein is characterized by greater substrate affinity (K_(M)) andhigher reaction velocity (V_(max)), as compared to those of mammaliancell-expressed recombinant human DNase I (e.g. Pulmozyme®). Methods formeasuring biological activity of DNase I are well known in the art, andinclude, inter alia, catalytic activity (e.g. methyl green assay,DNaseAlert™-based assay, fluorescence-based assay, as describedherewith), immunoreactivity with anti-DNase I antibody, Kunitz-basedhyperchromicity assay, assays measuring proton release upon DNAhydrolysis, which can be monitored by using chromophoric H+ ionindicators, and the like. In still other embodiments, the biologicalactivity of the plant-expressed recombinant human DNase I protein ischaracterized by specific activity (units DNase I catalytic activity permg enzyme protein) greater than that of mammalian cell-expressedrecombinant human DNase I (e.g. Pulmozyme®). In some embodiments, thespecific activity of plant-expressed recombinant human DNase I proteinis about 1.1, about 1.2, about 1.25, about 1.5, about 1.75, about 2.0,about 2.25, about 2.5, about 2.75, about 3.0, about 3.25, about 3.5,about 3.75, about 4.0, about 4.5 to 5.0 or more fold that of thespecific activity (e.g. U/mg) of mammalian cell-expressed recombinanthuman DNase I (e.g. Pulmozyme®). In some embodiments, the specificactivity of plant-expressed recombinant human DNase I protein is about3.0 to 3.5 fold that of the specific activity (e.g. U/mg) of mammaliancell-expressed recombinant human DNase I (e.g. Pulmozyme®).

As shown in Example 4 herein, when enzyme activity of DNase I proteinwas measured (see Table VIII) in the presence of increasingconcentrations of actin, greater resistance of the plant-expressedrecombinant human DNase I to inhibition of catalytic activity by actinwas observed, compared to that of mammalian cell-expressed recombinanthuman DNase I (e.g. Pulmozyme®). Thus, according to some embodiments theplant expressed recombinant human DNase I has greater resistance toactin inhibition of DNase I catalytic activity when compared to that ofmammalian cell-expressed recombinant human DNase I (e.g. Pulmozyme®). Insome embodiments, the inhibition of DNase I catalytic activity isexpressed as half-maximal inhibitory concentration of actin (IC₅₀, μgactin/ml), using, for example, the methyl green DNase I assay. Thus, insome embodiments, the IC₅₀ actin concentration for inhibition of theplant expressed recombinant human DNase I is at least 1.25, about 1.5,about 1.75, about 2.0, about 2.25, about 2.5, about 2.75, about 3.0,about 3.25, about 3.5, about 3.75, about 4.0, about 4.5 to 5.0 or more,or a range of about 1.5 to 2.5 fold greater than that of mammaliancell-expressed recombinant human DNase I (e.g. Pulmozyme®). In someembodiments, the IC₅₀ actin concentration for inhibition of the plantexpressed recombinant human DNase I activity is about 2.0 to 2.2 foldthat of mammalian cell-expressed recombinant human DNase I (e.g.Pulmozyme®).

As shown in Example 5, plant expressed recombinant human DNase Ieffectively reduces the rheological properties of sputum. When measuredin an in-vitro assay, incubation of the plant expressed recombinanthuman DNase I with samples of CF sputum results in reduction of theviscous modulus (as expressed by the loss modulus, G″) and reduction ofthe elasticity (as expressed by the storage modulus, G′) of the sputumsample (see FIGS. 12A-12D, 13A-13D, 14A-14B, 16 and 17). When comparedto the reduction of rheological parameters of sputum samples incubatedwith mammalian cell-expressed recombinant human DNase I (e.g.Pulmozyme®), the plant expressed recombinant human DNase I displayedgreater efficacy in reducing viscosity and elasticity than that ofmammalian cell-expressed recombinant human DNase I (e.g. Pulmozyme®)(FIGS. 12 and 13). Thus, according to some embodiments of the invention,the plant expressed recombinant human DNase I reduces viscous modulusand/or elastic modulus of sputum. In other embodiments, reduction inviscous and elastic modulus is expressed as G″ and G′, respectively. Inyet another embodiment, reduction of viscous and/or elastic modulus ofsputum by the plant expressed recombinant human DNase I is greater thanthat of mammalian cell-expressed recombinant human DNase I (e.g.Pulmozyme®) measured with in the same assay technique. Thus, in someembodiments, the plant expressed human recombinant DNase I of theinvention is biologically active, having catalytic activity, enzymekinetics and specific activity comparable or superior to that ofmammalian cell-expressed recombinant human DNase I, and effective inreducing rheological properties of CF sputum. In other embodiments, therheological properties of sputum are assayed using stress sweepmeasurements, and can be characterized by the cross-over points ofelastic and viscous stress values.

Thus, the human DNase I protein expressed in plant cells according tothe invention can be used to produce an inhalable dry powder formulationfor treatment or prevention of any condition or disease by pulmonaryadministration. According to some aspects of the invention, the drypowder formulation can be used for treatment or prevention ofmucus-associated conditions in a subject in need thereof. In someembodiments, the mucus associated conditions comprise respiratory orpulmonary disease or conditions.

In some embodiments of the invention, the dry powder formulationcomprising DNase I can be administered for reducing extracellular DNA ina secretion, fluid or tissue of a subject in need thereof. Secretions,tissues and fluids accessible to the dry powder formulations of theinvention include pulmonary secretions, fluids and tissues such as mucusand other extracellular brochipulmonary fluids, and secretions, fluidsand tissues accessible via the circulatory system, such as blood,intestinal mucosal secretions and the like.

Thus, according to some embodiments of the invention, there is provideda method for preventing or treating a pulmonary disease or conditionassociated with excess DNA in a pulmonary secretion in a subject in needthereof, the method comprising administering, via pulmonaryadministration, an effective amount of the dry powder inhalableformulation of the invention. Suitable formulations and dosage regimensare provided herewith in detail.

Respiratory conditions or diseases which can be treated byadministration of the dry powder formulation of the invention includeconditions associated with accumulation of mucus or other DNA containingsecretions or fluids, for example, in the airways. Such conditionsinclude, but are not limited to acute or chronic bronchopulmonarydisease, atelectasis due to tracheal or bronchial impaction andcomplications of tracheostomy chronic bronchitis, asthmatic bronchitis,cystic fibrosis, pneumonia, allergic diseases such as allergic asthma,non-allergic asthma, systemic lupus erythematosus, Sjogren's syndrome,bronchiectasis, emphysema, acute and chronic sinusitis, and even thecommon cold.

Non-respiratory conditions that can be treated by the dry powderformualtions of the invention include, but are not limited to, maleinfertility, metastatic cancer, viral, bacterial, fungal and protozoaninfections and sepsis, atherosclerosis, diabetes, delayed typehypersensitivity and uterine disorders.

In further embodiments, biological activity of the plant expressedrecombinant human DNase I of the invention is enhanced by the presenceof an additional pharmacological agent, for example, an agent whichreduces actin inhibition of DNase activity, such as one or moreinorganic salt selected from potassium, magnesium, calcium, zinc,lithium, manganese, cadmium, nickel, cobalt, ammonium, polyamine andmacrocyclic polyammonium salts. Agents suitable for combination with theplant expressed human recombinant DNase I of the invention, fortherapeutic applications such as treatment of pulmonary conditions (e.g.CF) are described in detail in U.S. Pat. No. 7,432,308 to Demeester etal., which is incorporated herein by reference in its entirety.

In some embodiments, combination of the plant expressed recombinanthuman DNase I with an additional pharmaceutical agent results inimprovement, and optionally synergic improvement in reduction ofrheological properties (e.g. viscous modulus and/or elastic modulus) ofsputum. In some embodiments, the additional agent is magnesium chlorideor magnesium sulfate.

In some embodiments of the invention, the dry powder formulationcomprising DNase I is combined with, or administered along with anadditional pharmaceutical agent, the additional pharmaceutical agentincluding, but not limited to one or more other pharmacologic agentsused to treat the conditions listed above, such as antibiotics,bronchodilators, anti-inflammatory agents, mucolytics (e.g.n-acetyl-cysteine), actin binding or actin severing proteins [e.g.,gelsolin; Matsudaira et al., Cell 54:139-140 (1988); Stossel, et al.,PCT Patent Publication No. WO 94/22465], protease inhibitors, or genetherapy product [e.g., comprising the cystic fibrosis transmembraneconductance regulator (CFTR) gene, Riordan, et al., Science245:1066-1073 (1989)]. Additional pharmaceutical agents can beadministered prior to, along with, subsequent to or in any othertemporal combination with the formulation of the invention. Regimen forcombination of the formulation of the invention with additional agentscan be formulated according to parameters such as specific conditions ordiseases, health status of the subject, methods and dose ofadministration, and the like. Determination of such combination regimencan be done, for example, by professionals such as attending physicians,hospital staff, and also according to predetermined protocols.

Herein the term “active ingredient” refers to at least the recombinanthuman DNase I protein (and optionally at least one of the abovepharmaceutical agents) accountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

As used herein, the term “subject in need thereof” refers to a subjectdiagnosed with or exhibiting one or more conditions associated with adisease or condition treatable by administration of DNase I, a subjectwho has been diagnosed with or exhibited one or more conditionstreatable by administration of DNase I in the past, or a subject who hasbeen deemed at risk of developing one or more conditions associated witha disease or condition treatable by administration of DNase I in thefuture due to hereditary or environmental factors. In certainembodiments of the invention, the subject in need thereof is sufferingfrom a disease or condition such as, but not limited to respiratoryand/or pulmonary disease or condition, male infertility, viralinfection, a uterine disorder, an endometrial disorder or condition,cancer, primary cancer and/or metastatic cancer.

In some embodiments, a subject in need thereof refers to a subject witha pulmonary condition having clinically abnormal spirometry values.Examples of spirometry parameters which can indicate the need of asubject include, but are not restricted to forced expiration volume₁(FEV₁), forced vital capacity (FVC), forced expiratory flow (FEF25-75)and the like. In some embodiments of the invention administration of theprhDNase to the subject results in an improvement in one or more of thespirometric parameters.

Pulmonary Administration of DNase

Pulmonary administration may be accomplished by suitable means known tothose in the art. Pulmonary administration of DNase requires dispensingof the biologically active substance from a delivery device into theoral cavity of a subject during inhalation. For purposes of the presentinvention, compositions comprising DNase are administered via inhalationof dry powder formulation of the invention, via a dry powder inhalerdelivery device. Such delivery devices are well known in the art andinclude, but are not limited to, metered dose and premetered dry powderinhalers, or any other appropriate delivery mechanisms that allow fordispensing of a solid or dry powder form.

Dry Powder Inhaler (DPI) Devices

According to some aspects of one embodiment, the dry powder formulationcomprising DNase, or biologically active portion thereof, is deliveredto a subject through a dry powder inhaler (DPI). A DPI is used todeliver an agent, such as DNase, in a solid or dry powder form using asubject's inspiration to deliver the dry powder to the lungs, instead ofa mist. A DPI is used to breathe in (inhale) the DNase so that it goesdirectly into the subject's lungs. A DPI is a propellant-free device,wherein the agent to be delivered is blended with suitable carriersknown in the art. The unit dose of agent used in a DPI device is often adry powder blister disc of hard capsule. A DPI produces dispersible andstable dry powder formulations which are inhaled, including spraydrying, spray-freeze drying, and micronized milling formulations. DPIdevices have been used to deliver macromolecular agents, includinginsulin, interferon (IFN), and growth hormone (GH). Examples of DPIdevices include, but are not limited to, the following:

The AIR® inhaler (Alkermes) which includes a small, breath-activatedsystem that delivers porous powder from a capsule (see WO 99/66903 andWO 00/10541). The porous particles have an aerodynamic diameter of 1-5urn and are prepared by spray drying. The AIR™ inhaler has been used todeliver albuterol, epinephrine, insulin, and hGH. The TurboHaler®(AstraZeneca) is also a DPI which may be used in the methods of theinvention and is described in EP patent 0799067, incorporated byreference herein. This DPI device is an inspiratory flow-driven,multidose dry-powder inhaler with a multi-dose reservoir that providesup to 200 doses of the drug formulation and dose ranges from a fewmicrograms to 0.5 mg. Examples of the TurboHaler™ include Pulmicort®(also Pulmicort® TurbuHaler®), Oxis® (formoterol) and Symbicort®(budesonide/formoterol).

Eclipse™ (Aventis) represents a breath actuated reusable capsule devicecapable of delivering up to 20 mg of formulation. The powder is suckedfrom the capsule into a vortex chamber where a rotating ball aids inpowder disaggregation as the subject inhales (see U.S. Pat. No.6,230,707 and WO9503846).

Another DPI device which may be used in the methods and compositions ofthe invention includes the Ultrahaler® (Aventis), as described in U.S.Pat. No. 5,678,538 and WO2004026380.

Another DPI device, which may be used in the methods and compositions ofthe invention includes the Bang Olufsen breath actuated inhaler, whichis a disposable breath actuated inhaler using blister strips with up tosixty doses (see EP 1522325).

An active DPI (also usable as an MDI—described below) described in WO94/19042 (Bespak) employs multiple, carbon fiber brush, setaceouselectrodes to disperse powders and aerosols into fine/particles/mists.As the patient inhales, 1 to 10 kvolts is passed through the electrodesto disperse the powder/aerosol. A breath sensor is employed to initiatethe electric discharge.

The HandiHaler® (Boehringer Ingelheim GmbH) is a single dose DPI device,which can deliver up to 30 mg of formulated drug in capsules (seeWO2004024156).

An example of this device is Spiriva® (tiotropium bromide). The PADD DPI(Britannia Pharmaceuticals) is a pressurized aerosol dry powder deliverydevice utilizing a novel formulation comprised of surface activephospholipids, dipalmitoyl phosphatidyl choline (DPPC) and phosphatidylglycerol (PG), prepared in the form of a fine powder. The PADD deviceoffers the highest payload possible with a propellant powered device,(see U.S. Pat. No. 6,482,391). Another DPI device, which may be used inthe methods and compositions of the invention includes the Pulvinal®inhaler (Chiesi) which is a breath-actuated multidose (100 doses) drypowder inhaler (see U.S. Pat. No. 5,351,683). The Pulvinal inhaler hasbeen used to deliver respiratory drugs such as salbutamol (Butovent®Pulvinal®), beclomethasone (Clenil® Pulvinal®) as well as budesonide andformoterol.

Another DPI device which may be used in the methods and compositions ofthe invention includes NEXT DPI™, which features multidose capabilities,moisture protection, dose counting and doses only when proper aspiratoryflow is reached (see EP1196146, U.S. Pat. No. 6,528,096, WO0178693,WO0053158).

The DirectHaler™ (Direct-Haler A/S) may also be used in the methods andcompositions of the invention (see U.S. Pat. No. 5,797,392). This singledose, premetered, pre-filled, disposable DPI device made frompolypropylene resembles a straw, and has been used to deliverformulations of budesonide and formoterol. The Accuhaler/Diskus™(GlaxoSmithKline) is a disposable small DPI device using doses in doublefoil blister strips (see GB2242134), which has been used to deliverflutacasone propionate/salmeterol xinafoate, flutacasone propionate,salmeterol xinafoate, and salbutamol.

In addition, the methods may include the FlowCaps® (Hovione), acapsule-based, re-fillable, reusable, pen-shaped, moisture-proof passivedry-powder inhaler (see U.S. Pat. No. 5,673,686).

In one embodiment, the DPI device used in the invention is a multi-dosedevice such as the Clickhaler® (Innovata PLC), (see U.S. Pat. No.5,437,270), used to treat asthma and COPD with a variety of drugs,including salbutamol (Asmasal®), beclomethasone (Asmabec®), andprocaterol hydrochloride (Meptin®) as well as budesonide and formoterol.Another DPI device suitable for use with the invention includes theDuohaler® (Innovata PLC) (see WO0139823). Duohaler® is actually ideallysuited for the delivery of fixed combination therapy with additionalcompositions/drugs for CF, asthma, COPD and the like.

In one embodiment, the DPI device used in the invention is an S2 unitdose (Innovata PLC), which is a re-useable or disposable single-dose DPIfor the delivery of a wide range of therapeutics in high concentrations(see AU3320101).

Yet another DPI device which may be used in the methods and compositionsof the invention includes Taifun® DPI (LAB International) which is amultiple-dose (up to 200) DPI device that is breath actuated and flowrate independent (see U.S. Pat. No. 6,132,394). In one embodiment, theDPI device used in the invention is MedTone® (Mannkind Corp., seeWO0107107) which comprises an intake section, a mixing section, and amouthpiece. The mouthpiece is connected by a swivel joint to the mixingsection. The intake chamber comprises a piston with a tapered piston rodand spring, and one or more bleedthrough orifices to modulate the flowof air through the device.

The mixing section holds a capsule with holes containing a dry powdermedicament, and further opens and closes the capsule when the intakesection is at a certain angle to the mouthpiece. The mixing section is aVenturi chamber to impart a cyclonic flow to air passing through themixing chamber. The mouthpiece includes a tongue depressor, and aprotrusion to contact the lips of the user to tell the user that the DPIis in the correct position. Technosphere® Insulin System, used for thetreatment of diabetes, consists of a dry-powder Technosphere®formulation (see US2004096403) of insulin and MedTone® inhaler throughwhich the powder is inhaled into the deep lung.

The powder formulation of the drug to be delivered in microparticles hasa size range between 0.5 and ten microns, preferably in the range of twoto five microns, formed of a material releasing drug at a pH of greaterthan 6.4.). In the Technosphere device, a dry powder insulin formulationcontaining insulin complexed to3,6-di(fumaryl4-aminobutyl)-2,5-diketopiperazine (hereinafter fumaryldiketopiperazine or FDKP) is used. The use of diketopiperazines for drugdelivery is known in the art (see for example U.S. Pat. No. 5,352,461;U.S. Pat. No. 5,503,852; U.S. Pat. No. 6,071,497; and U.S. Pat. No.6,331,318). Pulmonary drug delivery using diketopiperazine and othermicroparticles is disclosed in U.S. Pat. No. 6,428,771. Particularlyadvantageous devices for powder delivery are disclosed in U.S. Pat. No.7,464,706 and in U.S. Pat. No. 6,923,175.

Another DPI device which may be used in the methods and compositions ofthe invention includes Xcelovair™ (Meridica/Pfizer) which featurespre-metered, hermetically sealed doses in a fine particle fractiondelivery to achieve up to 50% fine particle mass.

Yet another DPI device which may be used in the methods and compositionsof the invention includes MicroDose® DPI (Microdose Technologies) whichis a small electronic DPI device that uses piezoelectric vibrator(ultrasonic frequencies) to deaggragate the drug powder (small or largemolecules, neat chemical or mixtures of drug and lactose up to 3 mgdrug) in an aluminum blister (single or multiple dose) (see U.S. Pat.No. 6,026,809).

In another embodiment, the DPI device used in the invention is NektarPulmonary Inhaler® (Nektar) which creates an aerosol cloud suitable fordeep lung delivery (see AU4090599, U.S. Pat. No. 5,740,794), usingcompressed gas to aerosolize the powder. The Nektar Pulmonary Inhaler®is used in Exubera® inhalable insulin (Pfizer, Sanofi-Aventis, andNektar), as well as to administer tobramycin, leuprolide, and singlechain antibodies.

Also included in the invention is the Nektar Dry Powder Inhaler®(Nektar) which is used in combination with Nektar Pulmonary Technology®(see US2003094173). The Nektar DPI is ideal for large payloads (2-50 mg)and a variety of molecular sizes, and has been used to delivertobramycin inhalation powder for lung infections in Cystic Fibrosis andamphotericin B for treatment of fungal infection. Also included in theinvention is the active DPI Oriel™ (see WO0168169).

In addition, EasyHaler® (Orion Pharma), a multidose dry powder inhalerfor lung and nasal delivery may be used in the methods and compositionsof the invention (see WO02102444). The EasyHaler® includes BeclometEasyHaler®/Atomide EasyHaler® (beclomethasone dipropionate) and BuventolEasyHaler®/Salbu EasyHaler® (salbutamol).

Also included in the invention is the Jethaler® (Pulmotec) whichutilizes the MAG (mechanical aerosol generation from a highly compressedsolid) technology for CFC-free dry-powder inhalation. The JetHaler® hasbeen used to deliver budesonide (Budesonidratiopharm@).

Yet another DPI device which may be used in the methods and compositionsof the invention includes AccuBreathe™ single dose DPI (Respirics) (seeWO03035137, U.S. Pat. No. 6,561,186). Also included in the invention isthe AcuBreather™ multidose DPI (Respirics) which uses an aclar/PVCmoisture protected blister cartridge capable of holding 25-50 mg ofpowder (30 dose and 15 dose devices respectively) and are capable ofholding and delivering two different drug formulations simultaneously(see U.S. Pat. No. 6,561,186), using i-Point™ technology for drugrelease.

Also included in the invention is the Twisthaler® (Schering-Plough),capable of 14-200 actuations (U.S. Pat. No. 5,829,434), packaged with adesiccant. Products including this DPI device include the AsmanexTwisthaler (mometasone furoate).

Another DPI device which may be used in the methods and compositions ofthe invention includes the multidose SkyeHaler® DPI (SkyePharma) (seeU.S. Pat. No. 6,182,655, WO97/20589), for dosing from 200 ug to 5 mg.This DPI is device is included in Foradil Certihaler® (formoterolfumarate). Also included in the invention is the refillable, multidoseNovolizer® (Meda AB) dry powder inhaler (U.S. Pat. No. 5,840,279, U.S.Pat. No. 6,071,498, WO9700703).

Another DPI device which may be used in the methods and compositions ofthe invention includes the Blister Inhaler™ (Meda AB), which is arefillable, multi-dose, breath activated, dry powder inhaler with dosecounter (U.S. Pat. No. 5,881,719, WO9702061), able to delivermoisture-sensitive compounds (e.g. proteins and peptides).

Other DPI devices include the SpinHaler® (Aventis and Rhone-PoulencRorer); the unit dose DPI (Bespak; a single unit dose device; see U.S.Pat. No. 6,945,953), the DiskHaler® (GlaxoSmithKline; a multidose devicefor local lung delivery—see U.S. Pat. No. 5,035,237), Rotohaler®(GlaxoSmithKline) (see U.S. Pat. No. 5,673,686, U.S. Pat. No.5,881,721); LABHaler® (LAB International; a breath-actuated disposablesingle dose dry powder delivery device); AirMaX™ (Ivax; a multiple dosereservoir inhaler; see U.S. Pat. No. 5,503,144); Aerolizer™ (Novartis);see U.S. Pat. No. 6,488,027, U.S. Pat. No. 3,991,761); Rexam DPI (RexamPharma; see U.S. Pat. No. 5,651,359 and EP0707862; bead inhaler multipledose (Valois; WO0035523, U.S. Pat. No. 6,056,169; a multiple dose DPIpulmonary delivery device on license from Elan/Dura/Quadrant); Aspirair®(Ventura; WO 02/089880; a single dose, breath activated DPI); andGyrohaler® (Ventura; GB2407042; a passive disposable DPI).

Other examples of commercially available dry powder inhalers suitablefor use in accordance with the methods herein include the Spinhaler®powder inhaler (Fisons) and the Ventolin® Rotahaler® (GlaxoSmithKline).See also the dry powder delivery devices described in WO 93/00951, WO96/09085, WO 96/32152, and U.S. Pat. Nos. 5,458,135, 5,785,049, and5,993,783, herein incorporated by reference.

In one embodiment, the invention provides a dry powder inhaler (DPI)device for pulmonary administration of DNase to a subject, wherein theDPI device comprises a reservoir comprising an inhalable powder or drypowder composition comprising the DNase, and a means for introducing theinhalable powder or dry powder composition into the subject viainhalation. The invention also provides an inhalable powder whichcomprises the DNase and is administered to the subject via a dry powderinhaler (DPI).

The DPI device used in the invention may be either a single dose or amultidose inhaler. In addition, the DPI device used in the invention mayalso be either pre-metered or device-metered.

Metered Dose Inhaler (MDI) Device

In one embodiment, the DNase, including an enzymatically active portionthereof, is delivered to a subject through metered dose inhaler (MDI)device. An MDI device uses a propellant to deliver reproducible metereddrug dose to the lung and/or airways, and comprises a drug or agent,propellants (e.g. hydrofluoroalkanes (HFA)), surfactants (e.g.phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol,lysophosphatidyl choline, phosphatidic acid, triglycerides,monogycerides, soy lecithin, fatty acids, and alkyl-polyglycosides), andsolvents. An MDI device is often a compact pressurized dispenser,including a canister, metering valve, and spacer. The dose administeredby an MDI device is generally in mg and ranges in volume from about 25to 100 mL. Additionally, MDI devices are advantageous as they aretamper-proof.

Examples of CFC-free MDI products include Albuterol® HFA (Ivax),Atrovent®-HFA (Boehringer-Ingelheim), Proventil®-HFA (3M), Flovent®-HFA(GSK), Qvar® (3M), Ventolin® HFA(GSK), Xopenex® HFA (3M/Sepracor),Salamol Easi-Breathe® CFC-Free (Ivax), Berotec® (Boehringer-Ingelheim),Berodual® (Boehringer-Ingelheim), Intal® Forte (Rhone/Aventis), andSeretide® EvoHaler® (GSK).

Examples of MDI devices include, but are not limited to, the following:

In one embodiment, the invention provides an MDI device for pulmonaryadministration of DNase to a subject, wherein the MDI device is anAutoHaler® (3M) (see U.S. Pat. No. 6,120,752). Examples of AutoHaler®devices being used to deliver therapeutic agents include Aerobid®(flunisolide), Alupent® (metaproterenol sulphate),Atrovent®/Atovent®-HFA (ipratropium bromide), Combivent® (albuterolsulfate/ipatropium bromide), MaxAir® AutoHaler® (pirbuterol acetate),Proventil®-HFA (albuterol sulphate), Qvar® (beclomethasone dipropionate)and Xopenex® HFA (levalbuterol hydrochloride).

Another MDI device which may be used in the methods and compositions ofthe invention includes the breath-activated MD Turbo™ (Accentia Bio),which transforms metered-dose inhalers into a breath-activated,dose-counting inhaler.

In one embodiment, the invention provides an MDI device for pulmonaryadministration of DNase to a subject, wherein the MDI device is thecontinuous inhalation flow device WatchHaler® (Activaero GmbH).

The portable drug delivery system EZ Spacer® (AirPharma) may also beused in the methods and compositions of the invention. In anotherembodiment, the Asmair® (Bang and Olufsen Medicom AS) MDI. In yetanother embodiment, the invention includes an Active DPI/MPI device(Bespak) (see WO9419042). In still another embodiment, the inventionprovides an MDI device for pulmonary administration of DNase to asubject, wherein the MDI device is a device for delivering meteredaerosols comprising an active ingredient in solution in a propellantconsisting of a hydrofluoroalkane (HFA) (see WO0149350; Chiesi).

Other examples of MDI devices which may be used in the invention includeMDI inhalers described in U.S. Pat. No. 6,170,717 (GlaxoSmithKline);EasiBreath® MDI (Ivax; W0193933, U.S. Pat. No. 5,447,150); MDI breathcoordinated inhaler and breath actuated inhaler (Kos; CA2298448 andWO2004082633); Tempo™ (MAP Pharma; U.S. Pat. No. 6,095,141, U.S. Pat.No. 6,026,808 and U.S. Pat. No. 6,367,471); Xcelovent™ (Meridica/Pfizer;WO9852634; a breath operated device that also has a dose counterfeature); and Increased dosage MDI (Nektar see WO2004041340; a devicecapable of delivering 2 mg to 5 mg of a formulated drug using HFApropellants) and a MDI described in WO03053501 (Vectura).

Thus, the invention also includes a metered dose inhaler (MDI) devicefor pulmonary administration of DNase to a subject, the MDI devicecomprising a pressurized canister comprising an aerosol comprising theDNase and a propellant, and a means for introducing the aerosol into thesubject via inhalation.

Formulations

DNase for use in the methods of the invention is formulated in drypowder formulation suitable for inhalation. Suitable preparationsinclude all dry powder formulation preparations so long as the particlescomprising the protein composition are delivered in a size rangeconsistent with that described for the delivery device, e.g., a drypowder form of the formulation.

Thus, a liquid formulation comprising DNase, or enzymatically activeportion thereof, intended for use in the methods of the presentinvention may either be used as a liquid solution or suspension in thedelivery device or first be processed into a dry powder form usinglyophilization or spray-drying techniques well known in the art. Powdercomprising a DNase such as a plant expressed recombinant human DNase I,may also be prepared using other methods known in the art, includingcrystallization or precipitation (see, for example, dry powdermicrospheres (PROMAXX; Baxter) described in U.S. Pat. No. 5,525,519;U.S. Pat. No. 5,599,719; U.S. Pat. No. 5,578,709; U.S. Pat. No.5,554,730; U.S. Pat. No. 6,090,925; U.S. Pat. No. 5,981,719; U.S. Pat.No. 6,458,387, each of which is incorporated herein by reference).

Where the liquid formulation is lyophilized prior to use in the deliverymethods of the invention, the lyophilized composition may be milled toobtain the finely divided dry powder consisting of particles within thedesired size range noted above. Where spray-drying is used to obtain adry powder form of the liquid formulation, the process is carried outunder conditions that result in a substantially amorphous finely divideddry powder consisting of particles within the desired size range notedabove. Similarly, if the starting formulation is already in alyophilized form, the composition can be milled to obtain the dry powderform for subsequent preparation as an aerosol or other preparationsuitable for pulmonary inhalation. Where the starting formulation is inits spray-dried form, the composition has preferably been prepared suchthat it is already in a dry powder form having the appropriate particlesize for dispensing as an aqueous or nonaqueous solution or suspensionor dry powder form in accordance with the pulmonary administrationmethods of the invention. For methods of preparing dry powder forms offormulations, see, for example, WO 96/32149, WO 97/41833, WO 98/29096,and U.S. Pat. Nos. 5,976,574, 5,985,248, and 6,001,336; hereinincorporated by reference.

The resulting dry powder form of the composition is then placed withinan appropriate delivery device for subsequent preparation as an aerosolor other suitable preparation that is delivered to the subject viapulmonary inhalation. Where the dry powder form of the formulation is tobe prepared and dispensed as an aqueous or nonaqueous solution orsuspension, a metered-dose inhaler, or other appropriate delivery deviceis used. A pharmaceutically effective amount of the dry powder form ofthe composition is administered in an aerosol or other preparationsuitable for pulmonary inhalation.

The amount of dry powder form of the composition placed within thedelivery device is sufficient to allow for delivery of apharmaceutically effective amount of the composition to the subject byinhalation. Thus, the amount of dry powder form to be placed in thedelivery device will compensate for possible losses to the device duringstorage and delivery of the dry powder form of the composition.Following placement of the dry powder form within a delivery device, theproperly sized particles as noted above are suspended in an aerosolpropellant. The pressurized nonaqueous suspension is then released fromthe delivery device into the air passage of the subject while inhaling.

The delivery device delivers, in a single or multiple fractional dose,by pulmonary inhalation a pharmaceutically effective amount of thecomposition to the subject's lungs. The aerosol propellant may be anyconventional material employed for this purpose, such as achlorofluorocarbon, a hydrochloro-fluorocarbon, a hydrofluorocarbon, ora hydrocarbon, including trichlorofluoromethane,dichlorodifluoro-methane, dichlorotetrafluoromethane,dichlorodifluoro-methane, dichlorotetrafluoroethanol, and1,1,1,2-tetra-fluoroethane, or combinations thereof. A surfactant may beadded to the formulation to reduce adhesion of the protein-containingdry powder to the walls of the delivery device from which the aerosol isdispensed. Suitable surfactants for this intended use include, but arenot limited to, sorbitan trioleate, soya lecithin, and oleic acid.

Devices suitable for pulmonary delivery of a dry powder form of aprotein composition as a nonaqueous suspension are commerciallyavailable. Examples of such devices include the Ventolin metered-doseinhaler (Glaxo Inc., Research Triangle Park, N.C.) and the Intal Inhaler(Fisons, Corp., Bedford, Mass.). See also the aerosol delivery devicesdescribed in U.S. Pat. Nos. 5,522,378, 5,775,320, 5,934,272 and5,960,792, herein incorporated by reference.

Where the solid or dry powder form of the formulation is to be deliveredas a dry powder form, a dry powder inhaler or other appropriate deliverydevice is preferably used. The dry powder form of the formulation ispreferably prepared as a dry powder aerosol by dispersion in a flowingair or other physiologically acceptable gas stream in a conventionalmanner. Examples of dry powder inhalers suitable for use in accordancewith the methods herein are described above.

When a formulation comprising a DNase is processed into a solid or drypowder form for subsequent delivery as an aerosol, it may be desirableto have carrier materials present that serve as a bulking agent orstabilizing agent. In this manner, the present invention disclosesstabilized lyophilized or spray-dried formulations comprising DNase foruse in the methods of the present invention. These compositions mayfurther comprise at least one bulking agent, at least one agent in anamount sufficient to stabilize the protein during the drying process, orboth. By “stabilized” is intended the DNase thereof retains itsmonomeric or multimeric form as well as its other key properties ofquality, purity, and potency following lyophilization or spray-drying toobtain the solid or dry powder form of the composition.

Preferred carrier materials for use as a bulking agent include glycine,mannitol, alanine, valine, or any combination thereof, most preferablyglycine. The bulking agent is present in the formulation in the range of0% to about 10% (w/v), depending upon the agent used. When the bulkingagent is glycine, it is present in the range of about 0% to about 4%,preferably about 0.25% to about 3.5%, more preferably about 0.5% to3.0%, even more preferably about 1.0% to about 2.5%, most preferablyabout 2.0%. When the bulking agent is mannitol, it is present in therange of about 0% to about 5.0%, preferably about 1.0% to about 4.5%,more preferably about 2.0% to about 4.0%, most preferably about 4.0%.When the bulking agent is alanine or valine, it is present in the rangeof about 0% to about 5.0%, preferably about 1.0% to about 4.0%, morepreferably about 1.5% to about 3.0%, most preferably about 2.0%.

Preferred carrier materials for use as a stabilizing agent include anysugar or sugar alcohol or any amino acid. Preferred sugars includesucrose, trehalose, raffinose, stachyose, sorbitol, glucose, lactose,dextrose or any combination thereof, preferably sucrose. When thestabilizing agent is a sugar, it is present in the range of about 0% toabout 9.0% (w/v), preferably about 0.5% to about 5.0%, more preferablyabout 1.0% to about 3.0%, most preferably about 1.0%. When thestabilizing agent is an amino acid, it is present in the range of about0% to about 1.0% (w/v), preferably about 0.3% to about 0.7%, mostpreferably about 0.5%. These stabilized lyophilized or spray-driedcompositions may optionally comprise methionine,ethylenediaminetetracetic acid (EDTA) or one of its salts such asdisodium EDTA or other chelating agent, which protect DNase againstmethionine oxidation.

Methionine is present in the stabilized lyophilized or spray-driedformulations at a concentration of about 0 to about 10.0 mM, preferablyabout 1.0 to about 9.0 mM, more preferably about 2.0 to about 8.0 mM,even more preferably about 3.0 to about 7.0 mM, still more preferablyabout 4.0 to about 6.0 mM, most preferably about 5.0 mM. EDTA is presentat a concentration of about 0 to about 10.0 mM, preferably about 0.2 mMto about 8.0 mM, more preferably about 0.5 mM to about 6.0 mM, even morepreferably about 0.7 mM to about 4.0 mM, still more preferably about 0.8mM to about 3.0 mM, even more preferably about 0.9 mM to about 2.0 mM,most preferably about 1.0 mM.

The composition of the invention can be formulated with additioningredients. These can contain any of the following ingredients, orcompounds of a similar nature: a binder such as microcrystallinecellulose, gum tragacanth or gelatin; an excipient such as starch orlactose, a disintegrating agent such as alginic acid, Primogel™, or cornstarch; a lubricant such as magnesium stearate or Sterotes™; a glidantsuch as colloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or orange flavoring. When the dosage unit form is a capsule, it cancontain, in addition to material of the above type, a liquid carrier. Inaddition, dosage unit forms can contain various other materials whichmodify the physical form of the dosage unit, for example, coatings ofsugar, shellac, or other enteric agents.

The stabilized lyophilized or spray-dried compositions may be formulatedusing a buffering agent, which maintains the pH of the formulationwithin an acceptable range when in a liquid phase, such as during theformulation process or following reconstitution of the dried form of thecomposition. In some embodiments the pH is in the range of about pH 4.0to about pH 8.5, about pH 4.5 to about pH 7.5, about pH 5.0 to about pH6.5, about pH 5.6 to about pH 6.3, and about pH 5.7 to about pH 6.2.Suitable pH's include about 4.0, about 4.5, about 5.0, about 5.1, about5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8,about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1,about 7.2, about 7.3, about 7.4, about 7.5, about 7.7, about 7.8, about7.9, about 8.0, about 8.2, about 8.4, about 8.6, about 8.8, about 9.0.In one particular embodiment, the pH is about 7.0 to 8.2. Suitablebuffering agents include, but are not limited to, citrate buffer,phosphate buffer, succinate buffer, more particularly a sodiumcitrate/citric acid. Alternatively imidazole or histidine or otherbase/acid that maintains pH in the range of about pH 4.0 to about 8.5can be used.

Buffers are chosen such that they are compatible with the drying processand do not affect the quality, purity, potency, and stability of theprotein during processing and upon storage.

Any of the formulations comprising human DNase I contemplated for use inthe methods of the invention may be formulated with at least onesurfactant. For pulmonary intracellular administration of the DNase, thesurfactant can be in an amount sufficient to enhance absorption of theinhaled particles comprising DNase to obtain an absorbable compositionfor use in pulmonary inhalation in accordance with the methods describedherein. Any surfactant that enhances absorption of a formulationcomprising DNase thereof in the manner disclosed herein may be used toobtain these absorbable protein-containing formulations. Surfactantssuitable for use in enhancing absorption of the inhaled DNase include,but are not limited to, polyoxyethylene sorbitol esters such aspolysorbate 80 (Tween 80) and polysorbate 20 (Tween 20);polyoxypropylene-polyoxyethylene esters such as Poloxamer 188;polyoxyethylene alcohols such as Brij 35; a mixture of polysorbatesurfactants with phospholipids such as phosphatidylcholine andderivatives (dipalmitoyl, dioleoyl, dimyristyl, or mixed derivativessuch as 1-palmitoyl, 2-olcoyl, etc.), dimyristolglycerol and othermembers of the phospholipid glycerol series; lysophosphatidylcholine andderivatives thereof; mixtures of polysorbates with lysolecithin orcholesterol; a mixture of polysorbate surfactants with sorbitansurfactants (such as sorbitan monoleate, dioleate, trioleate or othersfrom this class); poloxamer surfactants; bile salts and theirderivatives such as sodium cholate, sodium deoxycholate, sodiumglycodeoxycholate, sodium taurocholate, etc.; mixed micelles of DNasewith bile salts and phospholipids; Brij surfactants (such as Brij35-PEG923) lauryl alcohol, etc.). The amount of surfactant to be addedis in the range of about 0.005% to about 1.0% (w/v), preferably about0.005% to about 0.5%, more preferably about 0.01% to about 0.4%, evenmore preferably about 0.03% to about 0.3%, most preferably about 0.05%to about 0.2%.

The formulation of the invention may include a suitable dosage accordingto the disorder being treated. In one embodiment, the formulation of theinvention comprises a dose of about 0.01 mg to 10 mg of DNase orenzymatically active portion thereof.

Alternatively, the formulation of the invention comprises a dose ofabout 0.1 mg to 5 mg; about 1 mg to 5 mg; about 2.5 mg to 5 mg, about2.0 to 4.5 mg, about 2.2 to 4.0 mg, about 2.0 to 3.0 mg, about 2.2 to3.0 mg, about 2.3 to 3.0 mg, about 2.4 to 2.8 mg, about 2.4 to 2.6 mg;or about 2.5 mg of the DNase or enzymatically active portion thereof. Itis to be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that dosageranges set forth herein are exemplary only and are not intended to limitthe scope or practice of the claimed composition.

Thus, in some embodiments, the dosage regimen includes, but is notlimited to a single dose of the dry powder formulation of the invention,of 1.0 to 10 mg DNase I, administered daily, a single dose of 2.0 to 5mg DNase I, administered daily, a single dose of 2.0-3.0 mg DNase I,administered daily, a plurality of doses, each dose comprising 1.0-3.0mg DNase, the doses administered at least twice, 2-3 times, 2-4 times or2-6 times daily, a plurality of doses, each dose comprising 1.0-3.0 mgDNase, the doses administered once every 36 hours, once every 36-48hours, once every 36-72 hours, once every 2-3 days, once every 2-4 days,once every 2-5 days, or once every week, a plurality of doses, each dosecomprising 1.0-3.0 mg DNase, the doses administered once every 36 hours,once every 36-48 hours, once every 36-72 hours, once every 2-3 days,once every 2-4 days, once every 2-5 days, or once every week.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The formulation can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration before processing into adry powder. Sterile inhalable solutions can be prepared by incorporatingthe active compound (i.e., rhDNase, DNase I or other DNase) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged action of inhalable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

In one embodiment, the DNase or active portion for use in the methods ofthe invention is incorporated into a pharmaceutical formulation asdescribed in Examples 2-5. Supplementary active compounds can also beincorporated into the compositions for pulmonary delivery. In certainembodiments, a DNase or active portion for use in the methods of theinvention is coformulated with and/or coadministered with one or moreadditional therapeutic agents mentioned hereinabove. For example, DNaseI or active portion of the invention may be coformulated and/orcoadministered with one or more additional compositions that reduceactin inhibition (e.g. magnesium or potassium salts), and/or one or morechemical agents that inhibit mucus production (such as anti-inflammatoryagents, bronchodilators and/or mucus secretion blockers, as described inU.S. Pat. No. 7,763,610) or any combination thereof. Furthermore, theDNase of the invention may be used in combination with two or more ofthe foregoing therapeutic agents. Such combination therapies mayadvantageously utilize lower dosages of the administered therapeuticagents, thus avoiding possible side effects, complications or low levelof response by the patient associated with the various monotherapies.

The formulations of the invention may include a “therapeuticallyeffective amount” or a “prophylactically effective amount” of a DNaseprotein or active portion of the invention. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the DNase may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the DNase or active portion thereof toelicit a desired response in the individual. A therapeutically effectiveamount is also one in which any toxic or detrimental effects of theDNase are outweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

The invention also pertains to packaged formulations or kits forpulmonary administration of a DNase, e.g., DNase I. In one embodiment ofthe invention, the kit comprises a DNase, such as DNase I, andinstructions for pulmonary administration of the DNase, wherein theDNase is in a dry powder formulation suitable for inhalation.

The instructions may describe when, e.g., at day 1, day 4, week 0, week2, week 4, etc., the different doses of DNase shall be administered viainhalation to a subject for treatment.

Another aspect of the invention pertains to kits containing a dry powderformulation comprising a DNase, such as DNase I, and a pharmaceuticallyacceptable carrier, and one or more formulations each comprising anadditional therapeutic agent, and a pharmaceutically acceptable carrier.

The package or kit alternatively can contain the DNase and it can bepromoted for use, either within the package or through accompanyinginformation, for the uses or treatment of the disorders describedherein. The packaged formulations or kits further can include a secondagent (as described herein) packaged with or copromoted withinstructions for using the second agent with a first agent (as describedherein).

The term “tissue” refers to part of an organism consisting of cellsdesigned to perform a function or functions. Examples include, but arenot limited to, brain tissue, retina, skin tissue, hepatic tissue,pancreatic tissue, bone, cartilage, connective tissue, blood tissue,muscle tissue, cardiac tissue brain tissue, vascular tissue, renaltissue, pulmonary tissue, gonadal tissue, hematopoietic tissue.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose of the dry powderformulation can be formulated in animal models to achieve a desiredconcentration or titer. Such information can be used to more accuratelydetermine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually, for example, toprovide serum and cell levels of the active ingredient which aresufficient to induce or suppress the biological effect (minimaleffective concentration, MEC). The MEC will vary for each preparation,but can be estimated from in vitro data. Dosages necessary to achievethe MEC will depend on individual characteristics and route ofadministration.

Detection assays can be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of some embodiments of the invention may, if desired, bepresented in a pack or dispenser device, such as an FDA approved kit,which may contain one or more unit dosage forms containing the activeingredient. The pack may, for example, comprise metal or plastic foil,such as a blister pack. The pack or dispenser device may be accompaniedby instructions for administration. The pack or dispenser may also beaccommodated by a notice associated with the container in a formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the compositions or human or veterinaryadministration. Such notice, for example, may be of labeling approved bythe U.S. Food and Drug Administration for prescription drugs or of anapproved product insert. Compositions comprising a preparation of theinvention formulated in a compatible pharmaceutical carrier may also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition, as is further detailed above.

The formulation can be used for treatment or prevention of amucus-related respiratory condition in a subject in need thereof. Thus,according to another aspect of the present invention there is provided amethod for treating or preventing a respiratory condition such as cysticfibrosis (CF) or COPD in a subject in need thereof, the methodcomprising administering to the subject an effective amount of aformulation which includes, as an active ingredient thereof, a humanDNase I protein, and a pharmaceutical acceptable carrier. In someembodiments of the present invention, the human DNase I protein has anN-terminal Glycine residue. In other embodiments, the human DNase Iprotein has an amino acid sequence as set forth in SEQ ID NO: 5.

According to some embodiments of the present invention, the dry powderformulation comprising the human DNase I is administered by pulmonaryadministration, as detailed hereinabove.

In yet other embodiments, DNase can be used for treatment or preventionof male infertility, endometrial and uterine inflammatory disorders,viral infections and cancer, in particular, metastatic growth. In someembodiments, treatment of infertility, endometrial and uterineinflammatory disorders, viral infections and cancer metastases iseffected by administering to a subject in need thereof an effectiveamount of the dry powder formulation comprising the DNase I via a modeof pulmonary administration providing systemic delivery of the humanrecombinant DNase. In some embodiments, administering the dry powderformulation comprising the DNase I via pulmonary administration resultsin reduction in extracellular DNA in the subject, for example, of theextracellular DNA in a secretion, fluid or tissue of the subject. Insome embodiments, the extracellular DNA is pathological, and can beassociated with a disease or disorder from which the subject suffers.

The term “treating” refers to inhibiting, preventing or arresting thedevelopment of a pathology (disease, disorder or condition) and/orcausing the reduction, remission, or regression of a pathology. Those ofskill in the art will understand that various methodologies and assayscan be used to assess the development of a pathology, and similarly,various methodologies and assays may be used to assess the reduction,remission or regression of a pathology.

As used herein, the term “preventing” refers to keeping a disease,disorder or condition from occurring in a subject who may be at risk forthe disease, but has not yet been diagnosed as having the disease.

As used herein, the term “subject” includes mammals, preferably humanbeings at any age which suffer from the pathology. Preferably, this termencompasses individuals who are at risk to develop the pathology.

As used herein the phrase “treatment regimen” refers to a treatment planthat specifies the type of treatment, dosage, schedule and/or durationof a treatment provided to a subject in need thereof (e.g., a subjectdiagnosed with a pathology). The selected treatment regimen can be anaggressive one which is expected to result in the best clinical outcome(e.g., complete cure of the pathology) or a more moderate one which mayrelief symptoms of the pathology yet results in incomplete cure of thepathology. It will be appreciated that in certain cases the moreaggressive treatment regimen may be associated with some discomfort tothe subject or adverse side effects (e.g., a damage to healthy cells ortissue). The type of treatment can include a surgical intervention(e.g., removal of lesion, diseased cells, tissue, or organ), a cellreplacement therapy, an administration of a therapeutic drug (e.g.,receptor agonists, antagonists, hormones, chemotherapy agents) in alocal or a systemic mode, an exposure to radiation therapy using anexternal source (e.g., external beam) and/or an internal source (e.g.,brachytherapy) and/or any combination thereof. The dosage, schedule andduration of treatment can vary, depending on the severity of pathologyand the selected type of treatment, and those of skills in the art arecapable of adjusting the type of treatment with the dosage, schedule andduration of treatment.

It is expected that during the life of a patent maturing from thisapplication many relevant vectors, promoter elements, plant cells andcarriers will be developed and the scope of the terms provided herein isintended to include all such new technologies a priori.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1, 2, 317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein.

Other general references are provided throughout this document. Theprocedures therein are believed to be well known in the art and areprovided for the convenience of the reader. All the informationcontained therein is incorporated herein by reference.

Example 1 Construction of the Plant Deoxyribonuclease I ExpressionConstruct

cDNA encoding the human deoxyribonuclease I (DNase I) protein (EC3.1.21.1 GenBank: NM_(—)005223) was optimized and synthesized by GENEARTAG (Regensburg, Germany). The codon usage without the 22 amino acidleader peptide (e.g. endoplasmic reticulum target signal peptide) wasadapted to the codon bias of Nicotiana tabacum genes. During theoptimization process the following cis-acting sequence motifs wereavoided: Internal TATA-boxes, chi-sites and ribosomal entry sites,AT-rich or GC-rich sequence stretches, RNA instability elements (“killermotifs”), Repeat sequences and RNA secondary structures, splice donor(cryptic) and acceptor sites, branch points. In addition, regions ofvery high (>80%) or very low (<30%) GC content were avoided.

The nucleotide sequence of the native human DNase I leader peptide(endoplasmic reticulum target signal peptide) (FIG. 1, first 22 aminoacids in red) of the full length human DNase I protein (GenBank:NM_(—)005223) was replaced with a nucleotide sequence encoding the 33amino acid endoplasmic reticulum targeting signal peptide (leaderpeptide) of the Arabidopsis ABPI protein (marked in green in FIG. 1, SEQID NO: 4). This ER targeting signal peptide provides efficient targetingof DNase I to the secretory pathway and is then cleaved from thepolypeptide, by signal peptidase, once the protein has been translocatedinto the endoplasmic reticulum. In some cases the cleavage of ABPIsignal is not complete and the last (C-terminal) amino acid Glycine ofthe signal peptide remains, being added to the N terminal of the matureDNase I protein. As the optimized DNase I coding sequence does notcontain any compartment (organelle) localization signal at the 3′terminus, the recombinant DNase I, absent a C-terminal localizationsignal is directed to the plant cell apoplast (the extracellular spacesurrounding the plant cell symplast), and when expressed in plant cellcultures, is subsequently secreted into the culture medium. SEQ ID NO: 1represents the complete coding sequence of the expressed recombinanthuman DNase I polypeptide, including the N-terminal ABPI endoplasmicreticulum targeting signal peptide (SEQ ID NO: 4) and the human DNase Iprotein (SEQ ID NO: 6).

Example 2 Expression of Recombinant hDNase I in Plants

Transient Expression System in N. benthamiana

The use of plant viral vectors was chosen in this case as an alternativeto transgenic plants, allowing for the rapid, high level transientexpression of proteins in mature whole plants.

The protein of interest was expressed from a strong subgenomic viralcoat protein promoter. The system relies on transient amplification (byagroinfection) of viral vectors delivered to a plant by Agrobacterium,in which the plant functional promoter and the cDNA encoding a viralreplicon are transferred as T-DNA from Agrobacterium into plant cells.The T-DNA is transcribed in-planta by the plant promoter to generatebiologically active viral RNA that initiates self replication.

For the transient expression a 3-vector recombination system based onthe system previously developed as described (Gleba et al., Vaccine 232042-2048, 2005) was employed. DNase I cDNA was inserted into one of thevectors, and the two other vectors contained genes for construction ofthe whole viral replicon (RdRp and Integrase), thus generating abiologically active viral RNA capable of initiating self replication.

Transfection of Whole Plants—

N. Benthamiana plants were germinated and grown in commercial mix soil(Givaat Ada, Ill.) supplemented with granular slow release fertilizer(Scott Marysville, Ohio) under a long day (16 h light/8 h dark) lightregime at 24° C.-25° C.

Agrobacteria were transformed with the pICH20866-DNaseI based repliconvector system using electroporation (2500V, 5 msec) [den Dulk-Ra, A. andHooykaas, P. J. (1995) Methods Mol. Biol. 55:63-72]. Plants wereinfiltrated with Agrobacteria containing the 3 ICON plasmids by vacuuminfiltration with standard methods known in the art. Briefly, N.benthamiana plants 5-6 week old were infiltrated by immersing all aerialplant organs into a bacterial suspension and were placed in a vacuumchamber.

A minus (−) 0.8 bar vacuum was applied for 1 minute, followed by a quickreturn to atmospheric pressure. Plants were returned to the greenhousefor additional 5-7 days under the same growth conditions.

Screening for targeting to plant cell compartments was accomplishedusing the 3 vector recombination system described above, with the DNaseI-based vector including a sequence encoding an N-terminal ArabidopsisABPI signal peptide (marked in green in FIG. 1, SEQ ID NO: 4), followedby the nucleotide sequence of human DNase I, and further followed by oneof the below:

1) A nucleotide sequence (SEQ ID NO: 14) encoding the endoplasmicreticulum retention signal SEKDEL (SEQ ID 13), allowing retrieval of theexpressed protein from the Golgi apparatus, effectively maintaining theprotein in the endoplasmic reticulum;

2) A nucleotide sequence (SEQ ID NO: 16) encoding for the vacuoletargeting signal peptide GLLVDTM (SEQ ID 15), allowing transfer of theexpressed protein from the Golgi apparatus to the cell vacuole; or

3) No addition of any compartment localization signal at the 3′ terminusof the nucleic acid sequence, directing localization of the expressedDNase I protein to the plant cell apoplast, by default.

Tobacco Plant Extract:

Samples of Nicotiana benthamiana leaves were harvested 5 days postinfiltration and extracted in Laemmli buffer for SDS-PAGE, or inextraction buffer (25 mM HEPES-NaOH, 40 mM CaCl₂, 40 mM MgCl₂, pH 7.5)for assays of catalytic activity and ELISA of the plant expressedprotein.

Tobacco Plant Extract Purification:

Human DNase I protein from plant extracts was purified by centrifugationof the homogenized plant mass, discarding the plant debris. Thesupernatant was heated to 60° C. for 10 minutes and filtered through a 3μm filter, followed by filtration through a 0.65 μm filter. The filtratewas further purified on a hydrophobic interaction chromatography resin(Butyl 650C, Toyopearl) followed by further purification on an anionexchange chromatography resin (Poros 50HQ). The final material wasfiltered for sterility (0.2 μm).

Stable Expression in N. tabacum BY2 Cells

Agrobacterium mediated transformation is widely used to introduceforeign genes into a plant cell genome. This technique is based on thenatural capability of the agrobacterium to transform plant cells bytransferring a plasmid DNA segment, the transferred DNA (T-DNA), intothe host cell genome. Using this approach, a T-DNA molecule, consistingof a foreign gene and its regulatory elements, is randomly introducedinto the plant genome. The site of integration, as well as the copynumber of the gene insertions is not controlled, thus the transformationprocess results in a ‘pool’ of transgenic cells composed of cells withvarious levels of expression of the transgene.

The transgenic ‘pool’ is subsequently used for clone isolation. Cloneisolation results in the establishment of many single cell lines, fromwhich the clone with the highest expression level of the foreign gene isthen selected.

BY2 (Bright Yellow 2) suspension culture was co-cultivated, for 48hours, with the Agrobacterium tumefactiens EHA105 strain carrying thevector harboring the DNase I gene and the neomycin phosphotransferase(NPTII) selection gene.

Subsequently, the cells were kept in media supplemented with 50 mg/L ofKanamaycin and 250 mg/L Cefotaxime. The NPTII gene confers resistance toKanamycin, thus only NPTII positive BY2 cells survive in this selectionmedia. The Cefotaxime was used to selectively kill the agrobacterium,the plant cells being resistant to this antibiotic. Once a nicelygrowing transgenic cell suspension was established, it was used forscreening and isolating individual cell lines. To allow for theselection of individual cell lines, aliquots of highly diluted cellsuspension were spread on solid BY-2 medium. The cells were then grownuntil small calli developed. Each callus was then re-suspended in liquidculture. Media was then sampled and evaluated for DNase I levels. Thelines that secreted relatively high DNase I levels were then furtherre-analyzed and compared for DNase I levels ending with the finalselection of candidate DNase I expressing lines.

Media samples of transformed BY2 cells expressing the human DNase Iprotein were collected and when required, concentrated 5× by centrifugalfilters (Amicon Ultra, 10K, #UFC501096). DNase I catalytic activity incell's media was determined by DNA-Methyl Green assay and compared tototal DNase I amount, determined by Enzyme-linked immunosorbent assay(ELISA 1) as described hereinbelow.

Protein Purification from BY-2 Cells:

Recombinant human DNase-I protein secreted from tobacco suspension plantcells was purified by the following steps: at the end of thefermentation the intact tobacco cells were separated from the media byfiltration using 100 mesh filter bags. The cells were discarded and themedia containing the prhDNAse-I was collected for additional filtrationwith 0.2 μm filter sheets using filter-press apparatus. The DNase in thefiltrated media was further purified by two steps of chromatographycolumns of an anion exchange resin (Poros 50HQ, Applied Biosystems, USA)followed by hydrophobic interaction chromatography of Phenyl 650C resin(Toyopearl, Japan). The purified DNase collected from the last columnwas 0.2 μm filtrated and stored at 4° C.

Gel Electrophoresis:

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)separates proteins primarily by their molecular weight, proteinsmigrating as a linear function of the logarithm of their molecularweight in SDS-PAGE. For estimating the molecular weight of prh DNase I,migration on SDS-PAGE was compared to commercial molecular weightstandard proteins (New England BioLabs; cat No. P7708S) and to themigration of commercially available, mammalian-cell derived recombinanthuman DNase I, Pulmozyme® (Genentech, CA), expressed in CHO cells. prhDNase I and Pulmozyme were analyzed by 15% Tris-Glycine SDS-PAGE.Electrophoresis was performed using Criterion™ cell verticalelectrophoresis apparatus (Bio-Rad Lab.) with premixed electrophoresisTris-Glycine-SDS running buffer (Bio-Rad Laboratories). Fifteen percentacrylamide gels were prepared using premixed solutions of 40%Acrylamide/Bis and 10% SDS solution. Identification of the proteins wasperformed using two methods of detection: Coomassie brilliant bluestaining and Western blot analysis using specific antibodies andEnhanced Chemical Luminescence (ECL).

Coomassie Blue Staining

Following SDS-PAGE, gels were stained with Bio-Safe™ Coomasie Stain(Bio-Rad) according to manufacturer's instructions.

Western Blotting

Western Blot

Antibodies:

Detection of rh DNase1 was carried out by using either

(a) whole antiserum obtained from rabbits immunized against Pulmozyme®.These antibodies were prepared according to a standard polyclonalantibody preparation protocol including four immunizations with 3 mgantigen per rabbit, and collection of the serum after the fourthimmunization; or(b) affinity purified rabbit anti-prh DNase I and goat-anti-prh DNase Iacquired following immunization of rabbits and goats with prh DNase andaffinity purification on a Pulmozyme® column Thus, the purifiedantibodies detect the common backbone sequence of Pulmozyme andprhDNase. These antibodies were prepared by GenScript USA Inc inaccordance to GenScript standard polyclonal antibody preparationprotocol including four immunizations with 3 mg antigen per rabbit and 8mg antigen per goat, and collection of the serum after the forthimmunization.

PAGE and Blotting:

Following SDS-PAGE, proteins were transferred to a nitrocellulosemembrane using iBlot™ Dry Blotting System (Invitrogen). Transfer wasperformed at room temperature for 8 min. Then, the membrane was blockedwith 5% (v/w) non-fat dry milk powder in PBS supplemented with 0.05%(v/v) Tween-20, washed with PBS supplemented with 0.05% (v/v) Tween-20,bound to the primary antibody, followed by wash with PBS supplementedwith 0.05% (v/v) Tween-20 and incubation with secondary HRP-conjugatedantibody. Primary antibody was diluted 1:5000 (whole antiserum) or1:10000 (affinity purified rabbit and goat anti prh DNase I) in 1% (w/v)non-fat dry milk powder in PBS supplemented with 0.05% (v/v) Tween-20,and the secondary antibody-goat anti-rabbit or rabbit anti-goat HRPconjugated antibody (Jackson ImmunoResearch Laboratories, Inc., PA; catNo. 111-035-003 and cat No. 305-035-003 respectively) was diluted1:10,000 in 1% (w/v) non-fat dry milk powder in PBS supplemented with0.05% (v/v) Tween-20. Enhanced Chemi-Luminescence (ECL) reaction wasperformed with ECL detection kit (Pierce; cat. No. 34080). Theimmunoreactive bands were detected and documented using the MolecularImager Gel Doc XR System (Bio-Rad Laboratories, UK) for time intervalsup to 60 seconds, as needed.

Isoelectric Focusing

Isoelectric Focusing (IEF) separates proteins on the basis of theircharge in an electrical field. The protein migrates in a pH gradientgenerated by an electric field until it reaches a point in which its netcharge equals zero, and that pH reflects its isoelectric point (pI). Toidentify the pI of rh DNase I, protein was analyzed by polyacrylamideIEF gels using an XCell SureLock Electrophoresis Cell equipped with aPowerpac power supply (BIO-RAD) and by Imaged capillary isoelectricfocusing (iCE) using an iCE280 analyzer (Convergent Bioscience, TorontoCanada).

IEF analysis was performed using an XCell SureLock Electrophoresis Cellequipped with a Powerpac power supply (BIO-RAD). Pre-cast polyacrylamideIEF gels with a pH range of 3-7 were obtained from Invitrogen (Novex®,Invitrogen, cat No. EC6645BOX/EC66452BOX); buffers were obtained fromInvitrogen (anode buffer: Novex®, cat. No LC5300; cathode buffer:Novex®, cat. No. 5370; sample buffer: Novex®, cat. No. 5371); pI proteinstandards were obtained from SERVA (cat. No. 39212). Electrophoresisconditions: 100 mV-1 h, 200 mV-1 h, 500 mV-1.5 h. Bands were visualizedby Bio-Safe™ Coomassie Stain (Bio-Rad, cat No. 161-0787) according tothe manufacturer's instructions. The pI profile of rh DNase I (6μg/lane) was estimated using the pI protein standards, and then thebanding profile was compared to that of Pulmozyme®.

For imaged capillary isoelectric focusing analysis, a sample containedprh DNase I, pI markers 3.59 and 5.85 (protein simple, cat No. 102222and 102225, respectively), 0.35% methylcellulose (protein simple, catNo. 101876), and SERVALYT pH 3-7 (SERVA, cat No. 42945.01) wasintroduced to the capillary through an autosampler (PrinCE) and wasfocused in a capillary column under high voltage. The focusing processwas monitored in real-time using whole column imaging detection (WCID).

The resolved charge species appear as electrophoretic peaks that aredetected at a fixed wavelength of UV280 nm. pI values and peak areas foreach species were determined from the resultant electropherograms.

Mass Spectrometry

Molecular Weight Analysis

Molecular weight analysis was accomplished by mass spectrometry (MS) ofrh DNase I, using a matrix-assisted laser desorption ionizationtime-of-flight (MALDI-ToF) mass spectrometer and sinapinic acid as amatrix. The equipment was calibrated using molecular weight standards.About 2.5 micrograms of rh DNase were used for mass analysis.

Amino Acid Sequence and Structural Characterization

prh DNase I sequence was analysed using reverse-phase (RP-) HPLC coupledto mass spectrometry.

The protein samples were reduced using 2.8 mM DTT (60° C. for 30minutes), modified using 8.8 mM iodoacetamide in 100 mM ammoniumbicarbonate (in the dark, room temperature for 30 minutes) and digestedin 10% ACN and 10 mM ammonium bicarbonate with modified trypsin(Promega) or with chymotrypsin overnight at 37° C. in a 1:50enzyme-to-substrate ratio. 3% of the resulting peptides were resolved byreversed-phase liquid chromatography on a 0.075×200-mm fused silicacapillaries (J&W) packed with Reprosil reversed phase material (DrMaisch, GmbH, Germany).

Peptides were eluted with a linear 60 minutes gradient of 5 to 45%followed by 15 minutes at 95% acetonitrile with 0.1% formic acid inwater at flow rates of 0.25 μL/min. On-line mass spectrometry wasperformed by an ion-trap mass spectrometer (Orbitrap, Thermo) in apositive mode using repetitively full MS scan followed bycollision-induced dissociation (CID) of the 7 most dominant ionsselected from the first MS scan. The mass spectrometry data was analyzedusing the Sequest 3.31 software (J. Eng and J. Yates, University ofWashington and Finnigan, San Jose), compared to the human referencesequence.

Further sequencing and structural characterisation data was obtainedfrom trypsin digestion of prhDNase I, with or without a furtherdigestion with PNGaseA in the presence and absence of DTT and analysisby mass spectrometry

Disulfide Bonds and Free Sulfhydryl Content

Human DNase-1 sequence includes four cysteine residues and contains twodisulfides bonds. It was expected that the structure a recombinant humanDNase-1 expressed in plant cells (prhDNase I), will be similar to theauthentic human protein.

The assessment of the prhDNase I thiol content was obtained by using theEllman's method, which is a rapid and sensitive method for quantitativeanalysis of the total free thiols in peptides and proteins. The numberof free thiols found on prhDNase I also indicates the number of cysteineresidues that are engaged in disulfide bonds.

Free thiols analysis of prhDNase I and Pulmozyme® was carried outaccording to standard Ellman's method under denaturing conditions. Thereaction was performed by mixing the protein solution in sodiumphosphate buffer with 6M Guanidine Hydrochloride and the activesubstrate DTNB [5,5′-Dithiobis(2-nitrobenzoic acid)]. The solutions wereincubated at ambient conditions for 5-15 min During the reaction, TNB(2-nitro-5-thiobenzoic acid) was released. A reaction of 1 mole DTNBwith 1 mole thiol releases 1 mole of TNB anion with an extinctioncoefficient of 13,700 M⁻¹ cm⁻¹ at 412 nm. The number of thiols wascalculated as direct ratio between TNB concentration and prhDNase Iconcentration in the analyzed sample. A protein, containing free thiolswas used as positive control and DNase I formulation buffer (1 mM CaCl₂,150 mM NaCl, pH 6.1-6.5) was used as blank for analysis.

Glycan Structure of rh DNase

Glycan Analysis of the Plant-Expressed Human DNAseI:

Samples of recombinant human DNAseI protein product from the transformedcells were reduced with DTT alkylated with iodoacetamide, and separatedon a 12.5% SDS-PAGE. Bands corresponding to the correct molecular weightwere excised and subjected to glycan analysis consisting of trypsindigestion followed by peptide extraction and by both PNGase A and PNGaseF digestion. PNGase A digestion releases all the N-linked glycans andPNGase F release all glycans except those containing a 1-3 core fucose.

The free glycans were released, purified and then labeled with thefluorescent reagent anthranilamide (2AB) followed by removal of excess2AB.

Glycans were separated using a TSK gel Amide 80 normal phase HPLC anddetected using a fluorescence detector. A Dextran hydrolysate served asa ladder for calculation of glucose unit (GU) values. Glycan profile isconstructed by calculating the relative peak areas from the chromatogramof the PNGase A digestion. Assignment of the glycans is established bycalculation of the GU values of the peaks found in both endoglycosidedigestions and based on additional various exoglycoside digestions,comparison to known databases and confirmation of results by massspectrometric methods.

Reverse Phase-High Performance Liquid Chromatography (RP-HPLC)

RP-HPLC was used as an analysis method for the determination of purityof hr DNase I. The analysis employed a Jupiter 3μ, C18, 300 Å, 250×4.6mm column by Phenomenex. Analyses were performed on a Dionex, Ultimate3000 HPLC system equipped with a photodiode array detector.Chromatograms were routinely recorded at 2 wavelengths, 214 and 280 nm.

Elution Solvents:

Solvent A: 1 Liter, 0.1% TFA/H₂O (HPLC grade, J.T.Baker, cat #4218-03).Solvent A was prepared by adding 1.0 mL of TFA (Sigma, cat. # T6508) to0.999 liter of water (HPLC grade, J.T. Baker, cat. #4218-0.3).

Solvent B: 1 Liter, 0.1% TFA/Acetonitrile (HPLC grade, Sigma, cat.#34888). Solvent B was prepared by adding 1.0 mL TFA (Sigma, cat. #T6508) to 0.999 liter of acetonitrile (HPLC grade, Sigma, cat. #34888).

Chromatographic Method:

Chromatographic separations were performed on a Jupiter 3μ, C18, 300 Å,250×4.6 mm column (Phenomenex, cat. 00G-4263-E0). Chromatograms wereobtained using a linear gradient of 5% B for 3 min, followed by 5% B to43% B for 10 min, followed by 43% B to 55% B for 30 min, followed by 55%B to 95% B for 10 min followed by 95% B for 3 min followed by 95% B to5% B for 2 min, followed by 5% B for 21 min at a flow rate of 1 ml/minand at a temperature of 55° C. Each analysis sequence was commenced witha run of DNase I formulation buffer (1 mM CaCl₂.150 mM NaCl, pH 6.1-6.5)as a blank for rh DNase I.

Samples of prh DNase I (25 μg) in DNase I formulation buffer wereanalyzed. 25 μg of the rh DNase I was pipetted into a glass insert (250μL), which was inserted into an HPLC auto-sampler glass vial.

Results

Characterization of Plant-Expressed hr-DNase

Yields of hr DNase I expressed in plant cells of whole tobacco plantsvaried with targeting to different plant organelles. FIG. 2 shows thedifferences in yield of plant expressed human DNase I (relative to thatof apoplast targeting) when phr DNase I was expressed from a DNase Iconstruct encoding an N-terminal ER targeting signal peptide only (APO)directing the protein to the apoplast, from a DNase I construct encodingan N-terminal ER targeting signal peptide and a C-terminal vacuolartargeting signal peptide (VAC), or from a DNase I construct encoding anN-terminal ER targeting signal peptide and a C-terminal ER retentionsignal peptide, targeting the protein for retention in the ER (ER).Clearly, targeting to the apoplast results in the highest yield of thesethree expression options. Further, FIG. 2 also reveals the consistentcorrelation between levels of immunoreactive DNase I protein andcatalytic activity.

FIG. 3 shows an exemplary result of the migration of prh DNase I onSDS-PAGE (lanes 6-9), compared with mammalian cell expressed recombinanthuman DNase I (Pulmozyme®) (lanes 1-4). The plant expressed prh DNase Iconsists of one major band at a molecular weight of ˜30 KDa, migratingsimilarly that of the commercial preparation. The prh DNase Ipreparation did not contain any detectable contamination, indicating ahigh purity. An apparent slight difference in migration characteristicswas observed between the prh DNase I and the commercial preparation,with the plant expressed DNase I appearing to migrate faster. Withoutwishing to adhere to a single hypothesis, one possible explanation forsuch a difference in migration characteristics could be a difference inglycosylation patterns between the plant and mammalian cell-expressedenzymes. In order to compared for consistency between expression ofphrDNase I from plants and plant cell culture, 3 batches of prh DNase Iextracted from whole plants and 3 batches of prh DNase I extracted fromcultured cells were analyzed on SDS-PAGE, and were observed to have thesame, characteristic pattern of migration on the gel.

FIG. 4 shows an exemplary result of immunological cross reactivity ofthe prh DNase I separated on SDS-PAGE (lanes 6-9), compared with thecommercial, mammalian cell expressed recombinant human DNase I(Pulmozyme) (lanes 1-4), when reacted with unfractionated rabbitanti-human DNase I immune serum. The cross-reactive plant-expressed prhDNase I protein migrated as the 30 kDa molecular weight, similar to thecommercial preparation. Further, note that the plant-expressed prh DNaseI preparation does not contain any other detectable DNase I-relatedimmunoreactive species, indicating high purity of the preparation. Threebatches of DNase extracted from whole plant and 3 batches of DNaseextracted from cultured cells were analyzed and have shown the samemigrating characteristics. In addition, the same results were obtainedusing affinity purified rabbit anti-prh DNase

FIGS. 5A-5D shows a typical pI analysis of the plant-expressed prh DNaseI as compared to Pulmozyme®. The rh DNase I is characterized by 3 bands;two major isoforms ranged between 4.2-4.5 and a minor isoform with aslightly lower pI (FIG. 5A, lanes 2 and 3 and 5B, lane 2). This is incontrast to the commercial standard, Pulmozyme® (FIG. 5A, lane 1 and 5B,lane 3), which is characterized by a variety of isoforms ranging from pI3.5 to pI 4.5. Without wishing to adhere to a single hypothesis, onepossible explanation for such a difference in pI could be a differencein glycosylation patterns between the plant and mammalian cell-expressedenzymes. Three batches of DNase extracted from whole plant and 3 batchesof DNase extracted from cultured cells from cultures of differenttransformed BY2 lines were analyzed, and demonstrated the same migrationpattern upon pI analysis. pI values for each species were determinedfrom imaged capillary isoelectric focusing electropherograms (FIGS. 5Cand 5D). A main peak (MP, pI 4.41) and two acidic peaks (pre-MP1 andpre-MP2 with pI of 4.27 and 4.21) were observed, consistent with the pIprofile observed in the IEF gels (FIGS. 5A and 5B).

FIGS. 6A and 6B depict an analysis of the molecular weight ofplant-expressed prh DNase I by MALDI-ToF, showing the entire spectrumfrom 20000 to 180000 m/z (6A) or an enlargement of the prh DNase I peakat about 32000 m/z (6B). The molecular weight of the prh DNase I enzymeextracted from the plant cells was approximately 32,070 Da, while thetheoretical molecular weight of the prh DNase I polypeptide, based onthe proteins' 261 amino acid sequence, is 29,311 Da. Without wishing toadhere to a single hypothesis, one likely explanation for such adifference in the chromatographic characteristics is the difference inglycosylation patterns. Therefore, glycan structures may add theremaining ˜2760 Daltons. Three batches of prh DNase I extracted fromcultured cells were analyzed and have shown similar characteristics.

The difference in the molecular weight of the three phr DNase I mainisoforms seen in the MALDI analysis was approximately 200 Da. (FIG. 6B)This may correspond to a difference in glycan structures that can beattributed to one or two residues of N-Acetylglucosamine (203 Da). Thesame indication can be found in the variability of glycan structures.

Thus, the results of the mass spectrometry analysis confirm themolecular weight determination from the SDS-PAGE analysis (see FIG. 3),and both are consistent with a molecular weight of the prh DNase Icomparable to the calculated molecular weight of the expressedpolypeptide, and consistent with the molecular weight of mammalian cellexpressed recombinant human DNase I (Pulmozyme®).

Amino Acid Sequence of Plant-Expressed rh DNase I

Mass spectrometry analysis of the peptides generated by trypsin andchymotrypsin digestion of plant-expressed rh DNase I showed over 97.3%coverage of the 261 DNase I amino acid sequences that matched thepredicted amino acid sequence based on the DNA sequence of theexpression cassette (SEQ ID NO: 6). FIG. 7 represents the compositeamino acid sequence derived from the overlapping peptides, withunconfirmed amino acids in red, putative glycosylation sites bolded andunderlined, and the additional N-terminal glycine marked in green. Whencompared to the peptide sequence of the native human enzyme (SEQ ID NO:6, GenBank Accession No: NP_(—)005214.2) it is apparent that the aminoacid sequence of plant-expressed rh DNase I is identical to that ofcommercial, mammalian cell expressed dornase alfa (Pulmozyme®), and insome embodiments, with an additional glycine residue at position 1 atthe N terminus, derived from the ABPI signal peptide. Indeed, furtherHPLC and MS analysis of a trypsin digest and chymotrypsin sub-digest ofthe plant-expressed rh DNase I provided a full 261 amino acid sequence,confirming the accuracy of the sequence derived from overlappingpeptides (FIG. 7) and the identity of the amino acid sequence of theplant expressed rh DNase I and that of commercial, mammalian cellexpressed dornase alfa (Pulmozyme®), with the addition of a glycineresidue at position 1 at the N terminus, derived from the ABPI signalpeptide. Furthermore, the analysis of the trypsin digest of prhDNAse Itreated and not treated with PNGase A revealed the presence of glycansites on Asp 19 and Asp 107 as supported by the glycan analysis data inFIG. 8 (below). In addition, disulphide bridges were observed betweenCys102 and Cys105 and between Cys174 and Cys210.

The overlapping peptides are presented in the following coverage table(Table V), in which the first amino acid of plant-expressed rh DNase I,Gly, is numbered as amino acid 33, and where amino acids 1-32 arecomprised of the ABPI signal sequence (SEQ ID NO: 4), which are cleavedfrom the expressed polypeptide following transfer to the endoplasmicreticulum. Table Va shows peptides produced by trypsin and PNGasedigestion. In this table Gly, is denoted as amino acid number 1

TABLE VPeptides Identified Following Digestion with Trypsin and ChymotrypsinSEQ Position % Mass ID NO: Sequence 33-48 5.325060547  17GLKIAAFNIQTFGETK 34-39 2.029654905  18 LKIAAF 34-44 3.878161246  19LKIAAFNIQTF 34-48 5.150347722  20 LKIAAFNIQTFGETK 35-48 4.803860015  21KIAAFNIQTFGETK 36-42 2.378969069  22 IAAFNIQ 36-44 3.139192685  23IAAFNIQTF 36-45 3.313905509  24 IAAFNIQTFG 36-48 4.411379161  25IAAFNIQTFGETK 38-48 3.847234909  26 AFNIQTFGETK 39-48 3.629578364  27FNIQTFGETK 40-44 1.906776692  28 NIQTF 40-48 3.178963169  29 NIQTFGETK42-48 2.483049813  30 QTFGETK 43-48 2.090680443  31 TFGETK 49-573.019451292  32 MSNATLVSY 49-64 5.499783699  33 MSNATLVSYIVQILSR 53-644.264530922  34 TLVSYIVQILSR 55-64 3.608434794  35 VSYIVQILSR 58-642.538602758  36 IVQILSR 58-65 3.038226108  37 IVQILSRY 59-64 2.192115051 38 VQILSR 60-64 1.888571065  39 QILSR 63-74 4.439055196  40SRYDIALVQEVR 65-70 2.124510303  41 YDIALV 65-71 2.516879673  42 YDIALVQ65-73 3.215808036  43 YDIALVQEV 65-74 3.694099266  44 YDIALVQEVR 65-754.046539923  45 YDIALVQEVRD 65-78 5.079638575  46 YDIALVQEVRDSHL 65-836.477641206  47 YDIALVQEVRDSHLTAVGK 66-71 2.017256323  48 DIALVQ 66-743.194475917  49 DIALVQEVR 66-83 5.978017857  50 DIALVQEVRDSHLTAVGK 67-742.84203526   51 IALVQEVR 68-73 2.017256323  52 ALVQEV 68-74 2.495547553 53 ALVQEVR 69-74 2.277891008  54 LVQEVR 70-74 1.931403301  55 VQEVR72-83 4.019031885  56 EVRDSHLTAVGK 74-83 3.320103521  57 RDSHLTAVGK75-82 2.449331437  58 DSHLTAVG 75-83 2.841812291  59 DSHLTAVGK  75-10610.83964091  60 DSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGR 76-83 2.489371635  61SHLTAVGK 83-96 4.963013347  62 KLLDNLNQDAPDTY 84-89 2.149025427  63LLDNLN 84-90 2.541394797  64 LLDNLNQ 84-91 2.893835453  65 LLDNLNQD84-92 3.111491998  66 LLDNLNQDA 84-95 4.070909143  67 LLDNLNQDAPDT 84-964.570532493  68 LLDNLNQDAPDTY 84-97 4.990478738  69 LLDNLNQDAPDTYH 84-985.490102088  70 LLDNLNQDAPDTYHY  84-106 8.056098967  71LLDNLNQDAPDTYHYVVSEPLGR  84-110 9.564293519  72LLDNLNQDAPDTYHYVVSEPLGRNSYK  84-112 10.43796913  73LLDNLNQDAPDTYHYVVSEPLGRNSYKER 85-99 5.447158367  74 LDNLNQDAPDTYHYV86-96 3.877557079  75 DNLNQDAPDTY  86-106 7.363123553  76DNLNQDAPDTYHYVVSEPLGR  87-106 7.010682896  77 NLNQDAPDTYHYVVSEPLGR 88-106 6.661257247  78 LNQDAPDTYHYVVSEPLGR  90-106 5.965343891  79QDAPDTYHYVVSEPLGR  91-106 5.572974522  80 DAPDTYHYVVSEPLGR 92-982.654536986  81 APDTYHY  92-106 5.220533865  82 APDTYHYVVSEPLGR  93-1065.00287732   83 PDTYHYVVSEPLGR  94-106 4.705509252  84 DTYHYVVSEPLGR 95-106 4.353068596  85 TYHYVVSEPLGR  97-102 2.246976996  86 HYVVSE 97-104 2.890832771  87 HYVVSEPL  97-105 3.065545596  88 HYVVSEPLG 97-106 3.543836826  89 HYVVSEPLGR  97-107 3.893262475  90 HYVVSEPLGRN 97-110 5.052031378  91 HYVVSEPLGRNSYK  98-106 3.12389058   92 YVVSEPLGR 99-104 1.971263176  93 VVSEPL  99-107 2.97369288   94 VVSEPLGRN  99-1104.132461783  95 VVSEPLGRNSYK 111-115 2.22867221   96 ERYLF 113-1203.286263893  97 YLFVYRPD 113-122 3.982177249  98 YLFVYRPDQV 113-1234.248841949  99 YLFVYRPDQVS 113-124 4.466498494  10 YLFVYRPDQVSA 113-1265.122483137 101 YLFVYRPDQVSAVD 113-128 5.888771186 102 YLFVYRPDQVSAVDSY113-129 6.388394536 103 YLFVYRPDQVSAVDSYY 113-131 7.240458542 104YLFVYRPDQVSAVDSYYYD 113-132 7.592899198 105 YLFVYRPDQVSAVDSYYYDD 113-1337.767612023 106 YLFVYRPDQVSAVDSYYYDDG 114-128 5.389147837 107LFVYRPDQVSAVDSY 114-129 5.888771186 108 LFVYRPDQVSAVDSYY 115-1285.04266013  109 FVYRPDQVSAVDSY 115-129 5.542283479 110 FVYRPDQVSAVDSYY116-123 2.952115698 111 VYRPDQVS 116-125 3.473316229 112 VYRPDQVSAV116-126 3.825756886 113 VYRPDQVSAVD 116-128 4.592044935 114VYRPDQVSAVDSY 116-129 5.091668284 115 VYRPDQVSAVDSYY 116-130 5.591291634116 VYRPDQVSAVDSYYY 116-131 5.94373229  117 VYRPDQVSAVDSYYYD 116-1326.296172947 118 VYRPDQVSAVDSYYYDD 116-133 6.470885771 119VYRPDQVSAVDSYYYDDG 118-128 3.788877599 120 RPDQVSAVDSY 118-1294.288500948 121 RPDQVSAVDSYY 118-130 4.788124298 122 RPDQVSAVDSYYY118-132 5.493005611 123 RPDQVSAVDSYYYDD 140-150 4.037383416 124DTFNREPAIVR 141-150 3.684942759 125 TFNREPAIVR 143-150 2.924719144 126NREPAIVR 143-151 3.375334339 127 NREPAIVRF 144-150    2.575293495128REPAIVR 153-159 2.74066949  129 SRFTEVR 155-161 2.841713132 130 FTEVREF156-161 2.391097937 131 TEVREF 157-161 2.081489517 132 EVREF 160-1662.415813937 133 EFAIVPL 160-172 4.095594822 134 EFAIVPLHAAPGD 160-1754.834451899 135 EFAIVPLHAAPGDAVA 160-178 5.928764639 136EFAIVPLHAAPGDAVAEID 160-180 6.492908892 137 EFAIVPLHAAPGDAVAEIDAL160-181 6.992532241 138 EFAIVPLHAAPGDAVAEIDALY 160-182 7.344972898 139EFAIVPLHAAPGDAVAEIDALYD 160-183 7.648516884 140 EFAIVPLHAAPGDAVAEIDALYDV160-184 8.148140234 141 EFAIVPLHAAPGDAVAEIDALYDVY 160-190 10.33084718142 EFAIVPLHAAPGDAVAEIDALYDVYLDVQEK 162-178 5.082765067 143AIVPLHAAPGDAVAEID 162-180 5.64690932  144 AIVPLHAAPGDAVAEIDAL 162-1816.146532669 145 AIVPLHAAPGDAVAEIDALY 162-184 7.302140662 146AIVPLHAAPGDAVAEIDALYDVY 163-181 5.928876124 147 IVPLHAAPGDAVAEIDALY163-190 9.267191067 148 IVPLHAAPGDAVAEIDALYDVYLDVQEK 166-181 4.981476363149 LHAAPGDAVAEIDALY 167-180 4.135365306 150 HAAPGDAVAEIDAL 167-1814.634988656 151 HAAPGDAVAEIDALY 167-182 4.987429312 152 HAAPGDAVAEIDALYD167-183 5.290973298 153 HAAPGDAVAEIDALYDV 167-184 5.790596648 154HAAPGDAVAEIDALYDVY 167-190 7.973303599 155 HAAPGDAVAEIDALYDVYLDVQEK173-181 2.955207771 156 AVAEIDALY 173-190 6.293522714 157AVAEIDALYDVYLDVQEK 179-190 4.460352897 158 ALYDVYLDVQEK 180-1904.242696351 159 LYDVYLDVQEK 181-190 3.896208644 160 YDVYLDVQEK 182-1903.396585295 161 DVYLDVQEK 182-191 3.966729241 162 DVYLDVQEKW 184-1902.740600652 163 YLDVQEK 185-190 2.240977303 164 LDVQEK 185-1912.811121249 165 LDVQEKW 185-199 5.533190275 166 LDVQEKWGLEDVMLM 186-1901.894489595 167 DVQEK 186-191 2.464633542 168 DVQEKW 191-195 1.897439863169 WGLED 191-197 2.602489733 170 WGLEDVM 191-198 2.94897744  171WGLEDVML 191-199 3.350483324 172 WGLEDVMLM 191-200 3.525196149 173WGLEDVMLMG 191-208 6.151953511 174 WGLEDVMLMGDFNAGCSY 191-2106.933788727 175 WGLEDVMLMGDFNAGCSYVR 191-218 9.818443147 176WGLEDVMLMGDFNAGCSYVRPSQWSSIR 192-198 2.378833494 177 GLEDVML 192-1992.780339378 178 GLEDVMLM 192-208 5.581809564 179 GLEDVMLMGDFNAGCSY198-214 5.916115429 180 LMGDFNAGCSYVRPSQW 199-208 3.261246422 181MGDFNAGCSY 199-210 4.043081638 182 MGDFNAGCSYVR 199-214 5.569627722 183MGDFNAGCSYVRPSQW 199-218 6.927736058 184 MGDFNAGCSYVRPSQWSSIR 200-2082.859740538 185 GDFNAGCSY 200-210 3.641575754 186 GDFNAGCSYVR 200-2145.168121838 187 GDFNAGCSYVRPSQW 200-215 5.434786537 188 GDFNAGCSYVRPSQWS201-214 4.993409013 189 DFNAGCSYVRPSQW 203-214 4.190353162 190NAGCSYVRPSQW 204-214 3.840927513 191 AGCSYVRPSQW 207-214 3.132939701 192SYVRPSQW 207-218 4.491048037 193 SYVRPSQWSSIR 208-214 2.866275001 194YVRPSQW 209-214 2.366651651 195 VRPSQW 209-218 3.724759988 196VRPSQWSSIR 211-218 2.942924771 197 PSQWSSIR 213-218 2.378892004 198QWSSIR 215-220 2.333010341 199 SSIRLW 219-224 2.158151613 200 LWTSPT219-225 2.608766808 201 LWTSPTF 219-226 3.001136178 202 LWTSPTFQ 219-2273.571280124 203 LWTSPTFQW 219-228 3.917767831 204 LWTSPTFQWL 219-2314.914064263 205 LWTSPTFQWLIPD 219-234 5.750826164 206 LWTSPTFQWLIPDSAD219-241 7.924230704 207 LWTSPTFQWLIPDSADTTATPTH 219-244 8.957129041 208LWTSPTFQWLIPDSADTTATPTHCAY 219-246 9.787860928 209LWTSPTFQWLIPDSADTTATPTHCAYDR 220-246 9.441373221 210WTSPTFQWLIPDSADTTATPTHCAYDR 221-226 2.084504524 211 TSPTFQ 221-2272.654648471 212 TSPTFQW 221-228 3.001136178 213 TSPTFQWL 221-2417.007599051 214 TSPTFQWLIPDSADTTATPTH 221-244 8.040497388 215TSPTFQWLIPDSADTTATPTHCAY 221-246 8.871229275 216TSPTFQWLIPDSADTTATPTHCAYDR 225-246 7.687979666 217FQWLIPDSADTTATPTHCAYDR 226-244 6.406632585 218 QWLIPDSADTTATPTHCAY226-246 7.237364471 219 QWLIPDSADTTATPTHCAYDR 228-246 6.274851155 220LIPDSADTTATPTHCAYDR 229-244 5.097631562 221 IPDSADTTATPTHCAY 229-2465.928363448 222 IPDSADTTATPTHCAYDR 232-246 4.932067017 223SADTTATPTHCAYDR 234-246 4.447745772 224 DTTATPTHCAYDR 235-2443.264573229 225 TTATPTHCAY 236-246 3.785696695 226 TATPTHCAYDR 239-2462.948823309 227 PTHCAYDR 245-250 2.060234463 228 DRIVVA 245-2522.636453171 229 DRIVVAGM 245-253 2.982940878 230 DRIVVAGML 245-2543.329428586 231 DRIVVAGMLL 245-255 3.807719815 232 DRIVVAGMLLR 247-2532.152208992 233 IVVAGML 247-254 2.498696699 234 IVVAGMLL 247-2552.976987929 235 IVVAGMLLR 247-256 3.151700754 236 IVVAGMLLRG 248-2552.630500222 237 VVAGMLLR 249-255 2.326956236 238 VAGMLLR 250-2552.023412249 239 AGMLLR 252-259 2.630500222 240 MLLRGAVV 254-2664.11110757  241 LRGAVVPDSALPF 255-266 3.764619863 242 RGAVVPDSALPF256-262 1.974201118 243 GAVVPDS 256-263 2.191857663 244 GAVVPDSA 256-2642.53834537  245 GAVVPDSAL 256-265 2.835713438 246 GAVVPDSALP 256-2684.086369477 247 GAVVPDSALPFNF 256-271 4.914051937 248 GAVVPDSALPFNFQAA256-272 5.413675286 249 GAVVPDSALPFNFQAAY 256-273 5.588388111 250GAVVPDSALPFNFQAAYG 256-274 5.934875818 251 GAVVPDSALPFNFQAAYGL 256-2766.553981174 252 GAVVPDSALPFNFQAAYGLSD 256-293 12.44599823 253GAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK 267-272 2.185617005 254 NFQAAY267-273 2.360329829 255 NFQAAYG 267-286 6.777625088 256NFQAAYGLSDQLAQAISDHY 267-293 9.217939951 257 NFQAAYGLSDQLAQAISDHYPVEVMLK268-293 8.868514302 258 FQAAYGLSDQLAQAISDHYPVEVMLK 269-293 8.417899107259 QAAYGLSDQLAQAISDHYPVEVMLK 270-293 8.025529737 260AAYGLSDQLAQAISDHYPVEVMLK 271-293 7.807873192 261 AYGLSDQLAQAISDHYPVEVMLK273-280 2.547459231 262 GLSDQLAQ 273-282 3.111603483 263 GLSDQLAQAI273-285 4.150655085 264 GLSDQLAQAISDH 273-289 5.646574866 265GLSDQLAQAISDHYPVE 273-290 5.950118852 266 GLSDQLAQAISDHYPVEV 273-2916.351624736 267 GLSDQLAQAISDHYPVEVM 273-292 6.698112443 268GLSDQLAQAISDHYPVEVML 273-293 7.090593298 269 GLSDQLAQAISDHYPVEVMLK275-293 6.569392766 270 SDQLAQAISDHYPVEVMLK 277-293 5.95028741  271QLAQAISDHYPVEVMLK 279-293 5.211430333 272 AQAISDHYPVEVMLK 281-2934.601404418 273 AISDHYPVEVMLK 283-293 4.037260166 274 SDHYPVEVMLK285-293 3.41815481  275 HYPVEVMLK 287-293 2.498585215 276 PVEVMLK

TABLE Va Peptides Identified Following Digestion with Trypsin and PNGaseSEQ Position ID NO: Sequence  4-16 278 IAAFNIQTFGETK 17-32 279MSNATLVSYIVQILSR 33-42 280 YDIALVQEVR 43-51 281 DSHLTAVGK 52-74 282LLDNLNQDAPDTYHYVVSEPLGR 52-78 283 LLDNLNQDAPDTYHYVVSEPLGRNSYK  81-112284 YLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNR  81-118 285YLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNR EPAIVR 113-118 286 EPAIVR 119-122 287FFSR 123-127 288 FTEVR 128-158 289 EFAIVPLHAAPGDAVAEIDALYDVYLDVQEK215-223 290 IVVAGMLLR 224-261 291 GAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK

Free Sulfhydryl Content (Ellman's Method)

Quantitative assay of the free-thiols indicated that both phr DNase Iprotein and Pulmozyme® lack free sulfhydryls, while the positive controldetected four free sulfhydryls (thiols) as expected. These resultsconfirm that in phr DNase I four Cysteine amino acids are bound in 2disulfide bonds (Cys 102-Cys 105 and Cys 174-Cys210) as was suggestedfor DNase-1 and Pulmozyme®.

Glycan Analysis:

Preliminary analysis was performed to characterize the various glycanstructures of prhDNase I produced from BY2 cell culture. FIGS. 8A-8Cshow NP-HPLC profile of glycans derived from three separate batches ofprhDNase I using PNGase A to release the glycans. Release of glycans byPNGase F indicated a low abundance of glycans without core fucosestructures in prhDNase I. The glycan profile of all three batches ofprhDNase I indicated a similar profile. Peaks in the profiles shown inFIG. 8A correlate to the detailed structures in FIG. 8B indicating therelative amounts of glycans released from the batches of prhDNase I.

The glycan analysis of recombinant human DNAseI protein product from thetransformed tobacco cells revealed a glycan profile comprising two mainglycan peaks consisting of at least four glycan structures (e.g. peaks3+4 and 5). Predominant glycan structures representing a high percentageof the glycan profile (over 80%) contain mannose 3-β-(1,2)xylose-α-(1,3) fucose [Fc(3)M3X] and/or mannose 4-α-(1,2) xylose (M4X)with an additional one or two N-acetylglucosamine substitution on theouter mannose sugars (FIG. 8B). The other glycans contain smallerstructures missing either fucose or N-acetylglucosamine substitutions(3-6%) or hybrid structures (up to 14%).

FIG. 8C shows a comparison of glycans found in prhDNase I released byPNGase A compared to glycans released from Pulmozyme® by PNGase F. Thewide variations in glycosylation pattern found in Pulmozyme® are due tothe abundance of bi- and tri-antennary glycans with additional sialicacid residues attached, leading to a mixture of charged and unchargedglycan moieties. Clearly, phr DNase I glycosylation is more homogeneousand does not include charged glycans.

Purity of Plant-Expressed Rh DNase Preparation:

FIGS. 8A-8D represent a typical chromatogram of the plant-expressed prhDNase I, analyzed by RP-HPLC at 214 nm (9A, 9B) and at 280 nm (9C, 9D),after subtraction of start-of-sequence blank. Insets 9B and 9D areexpanded views of the prh DNase I peak. In both chromatograms, the prhDNase I resolves as a main peak (>93.6% purity) with a retention time of28.3 minutes and an additional two small peaks, a pattern characteristicof all batches of the prh DNase I tested.

Thus, the prh DNase I preparation is characterized by molecular weight,pI, free sulfhydryl content, immune cross-reactivity and amino acidsequence comparable to but distinct from that of commercially availablemammalian cell expressed DNase I, and consistent with the translationproduct of the expressed human DNase I coding sequence of SEQ ID NO: 9.Further, the plant-expressed rh DNase I is secreted into the medium ofcells in suspension culture, and can be purified to a high degree ofpurity, containing less than 7% impurities detected by HPLC at both 214and 280 nm.

Example 3 Biological Activity of Plant-Expressed rh Dnase I

Biological activity of the plant-expressed rh DNase I was assayed usingseveral assays, and compared to that of the mammalian-cell expressedcommercially available human recombinant enzyme Pulmozyme® (Genentech,MA).

Methods:

DNA-Methyl Green Assay

Deoxyribonuclease I (DNase I) is an endonuclease that cleaves DNApreferentially at phosphodiester linkages, yielding5′-phosphate-terminated polynucleotides with a free hydroxyl group onposition 3′, on average producing tetranucleotides.

Activity of DNase I was assessed by the Methyl Green enzymatic activityassay, employing DNA from Salmon Testis (Sigma cat No. D1626) complexedwith the dye methyl green (Sigma cat No. M8884) as a substrate. Methylgreen intercalates between the stacked bases of double-stranded DNA.Once the long DNA molecules are hydrolyzed into tetranucleotides as aresult of DNase1 activity, dissociation of methyl green from the DNAoccurs, the free methyl green decolorizing in a second, nonenzymaticreaction. The spontaneous decolorization of free methyl green at neutralpH is likely to result from tautomerization of the dye.

Standard curves were prepared by dilution of purified standard phrDNaseI in activity Buffer (25 mM HEPES-NaOH, 4 mM CaCl₂, 4 mM MgCl2, 0.1%BSA, 0.05% Tween-20, pH 7.5) at concentrations ranging from 0.3 to 20ng/ml at 2-fold series dilutions. Samples and controls were prepared ina similar matter. One hundred microliters of standards, controls andsamples was added in duplicates to a 96-well plate (NUNC) containing 100μl of DNA-methyl green substrate and the contents were mixed thoroughly.The plates were then sealed and agitated for 30 min at room temperature,then incubated overnight at 37° C. and absorbance measured at 620 nm.Absorbance was plotted versus standards concentrations and the data werefit to a 4-parameter logistic model by the nonlinear regression methodof Marquardt.

DNase I activity in different expression systems was expressed aspercent of the activity measured in the apoplast targeted cells (100%).(see FIG. 2)

Enzyme-Linked Immunosorbent Assay (ELISA)

Enzyme-linked immunosorbent assay (ELISA) was used to detect thepresence of immunoreactive rh Dnase I in a sample. Two different ELISAmethods were performed. The first ELISA assay (ELISA 1) was developedusing antibodies acquired against Pulmozyme®. The second ELISA assay(ELISA 2) was developed using antibodies acquired against plant derivedDNase I.

The total content of prh DNase I was assessed by indirect sandwich ELISAassays. The antibodies used in ELISA 1 include unfractionated rabbitanti-human DNase I antiserum and chicken yolk IgY fraction. Theseantibodies were prepared following immunization of rabbits and chickenswith commercially available, mammalian cell expressed human DNase I(Pulmozyme®). Unfractionated rabbit anti-human DNase I antiserum wasprepared in accordance with a standard polyclonal antibody preparationprotocol including four immunizations with 3 mg antigen per rabbit, andcollection of the serum after the fourth immunization. Anti-human DNaseI yolk IgY fraction was prepared in accordance to a standard polyclonalantibody preparation protocol including four immunizations with 2.5 mgantigen per chicken, and collection of the yolks after the fourthimmunization.

The antibodies used in ELISA 2 included rabbit anti-prh DNase I andgoat-anti-prh DNase I. These antibodies were prepared by immunization ofrabbits and goats with prh DNase and affinity purification on aPulmozyme® column Thus, the purified antibodies detect/bind the commonbackbone sequence of Pulmozyme® and prh DNase I. The antibodies wereprepared by GenScript USA Inc in accordance to GenScript standardpolyclonal antibody preparation protocol including four immunizationswith 3 mg antigen per rabbit and 8 mg antigen per goat, and collectionof the serum after the forth immunization.

Microtiter 96-well plates (Costar cat No. 9018 for ELISA 1 and NUNC catNo. 442404 for ELISA 2) were incubated for overnight at 4° C. with 100μl/well of whole rabbit antiserum 2500-fold diluted (ELISA 1) or 1 ug/mlgoat anti-prh DNase I antibody (ELISA 2) and diluted incarbonate-bicarbonate buffer pH 9.6 (Sigma cat No. C3041).

Plates were then washed four times with wash buffer prepared accordingto manufacturer's instructions (KPL cat No. 50-63-00), blocked with 300μl of Blocker Casein™ (Pierce cat No. 37528), incubated for one hour at25° C. and washed again four times with wash buffer. A standard curvewas prepared by dilution of standard prh DNase I in dilution buffer (1%(w/v) BSA in phosphate buffered saline) at concentrations ranging from0.3125 to 20 ng/ml (ELISA 1) or 0.1562 to 10 ng/ml (ELISA 2) at 2-foldseries dilutions. Similarly, samples and controls were diluted 100- to100,000-fold in dilution buffer. One hundred microliters of standards,controls and samples were added in duplicates to the wells of pre-coatedand pre-blocked plates, and agitated for two hours at 25° C. orincubated overnight at 4° C. Plates were then washed 4 times andincubated for 1.5 hours at 25° C. with 100 μl/well of chicken yolk IgYfraction diluted 20,000-fold in the dilution buffer (ELISA 1) or rabbitanti-prh DNase I antibody diluted 10,000-fold in the dilution buffer.Following four additional washing steps, 100 μl of donkey anti-chickenHRP conjugated antibody (Jackson ImmunoResearch Laboratories, Inc., PA;cat No. 703-035-155) (ELISA 1) or donkey anti-rabbit HRP conjugatedantibody (Jackson ImmunoResearch Laboratories, Inc., PA; cat No.711-035-152) (ELISA 2), diluted 5000-fold in dilution buffer, was addedand plates were incubated for one hour at 25° C. Next, plates werewashed extensively and supplemented with 100 μl TMB solution (Millipore;cat No. ES001). Upon color development reaction was stopped by additionof 100 μl of 10% (v/v) HCl and then read at 450 nm. Absorbance wasplotted versus the standards concentrations and data were fit to a4-parameter logistic model by the nonlinear regression method ofMarquardt.

DNase I immunoreactivity in different expression systems was expressedas percent of the activity measured in the apoplast targeted cells(100%)(see FIG. 2).

Enzyme Kinetics:

The kinetics of an enzymatic reaction are typically studied by varyingthe concentration of substrate and plotting the rate of productformation as a function of substrate concentration (velocity). In theconventional case, this yields a typical hyperbolic Michaelis-Mentencurve and a linear reciprocal Lineweaver-Burk plot, from which thekinetic constants of the enzyme can be calculated. For enzyme reactionsexhibiting simple Michaelis-Menten kinetics, the Michaelis constant(K_(M)), or the substrate concentration at half maximum velocity(V_(max)/2) represents the dissociation constant of the enzyme-substrate(ES) complex or the affinity for substrate. Low K_(M) values indicatehigher substrate affinity.

The kinetic constant (k₂), often called k_(cat), represents the numberof substrate molecules converted into product per unit time at a singlecatalytic site when the enzyme is fully saturated with substrate.k_(cat) is usually the reaction rate limiting step and is calculated bydividing V_(max) by the enzyme's concentration. k_(cat)/K_(M) is oftenthought of as a measure of enzyme efficiency.

Many enzymes do not behave in a conventional way, their velocity curvesrising to a maximum and then declining as the substrate concentrationrises, referred to as substrate inhibition, and thought to be the resultof binding of more than one substrate molecule to an active site.Substrate inhibition is considered a biologically relevant regulatorymechanism although it is often observed when using artificially highsubstrate concentrations, in a laboratory setting.

Kinetic plots for substrate inhibition represent variation of initialvelocity as a function of substrate concentration or asdouble-reciprocal plot of the variation of initial velocity withsubstrate concentration. Since the same compound acts at the same timeas both substrate and inhibitor the double reciprocal plot does notyield a straight line. It may be assumed that inhibitory effect isnegligible at low substrate concentration and from an asymptote to thecurve in this region K_(M) and V_(max) can be estimated.

Assays of the enzyme kinetics of DNase I activity were performed usingthe DNaseAlert™ (custom made by IDT) fluorescence-quenchedoligonucleotide probe, which has a HEX™ reporter dye(hexachlorofluorescein) on one end and a dark quencher on the other end,and which emits a detectable fluorescent signal upon nucleasedegradation. The DNA sequence has been carefully optimized to react witha wide variety of nucleases; it contains domains that will react withsingle-stranded endonucleases, certain single-stranded exonucleases, anddouble-stranded nucleases.

The intact substrate has little or no fluorescence, but when cleaved bya DNase, the hydrolyzed substrate fluoresces pink (536 nm or UVexcitation, 556 nm emission) when cleaved, and can be detected visuallyor using a fluorometer. Plots of DNase I initial velocity versussubstrate concentration display a velocity curve that rises to a maximumand then declines as the substrate concentration increases (FIG. 10A).This pattern can characterize a typical substrate inhibition behavior.The double reciprocal plot allows the extraction of K_(M) and V_(max)values by abscissa intercept with an asymptote to the high substrateregion (approaching the origin), where the inhibitory effectpredominates (FIG. 10B).

DNaseAlert™ Based Kinetic Assay

DNaseAlert™ substrate (IDT; custom made) was diluted in Activity Buffer(25 mM HEPES-NaOH, 4 mM CaCl₂, 4 mM MgCl₂, 0.1% BSA, 0.05% Tween-20, pH7.5) at concentrations ranging from 1 to 40 μM. Ten microliters ofsubstrate was divided in duplicates into a black 96-well plate (Greinercat No. 655900) and the reaction was initiated by rapid addition of 10ul DNase I to a final concentration of 50 ng/mL. The plate wasimmediately incubated in a fluorometer and real-time data was collected(535 nm excitation, 565 nm emission) at 30 seconds intervals for 3minutes.

Initial velocities were extracted by using fluorescent product standardcurve. Product standard curve was prepared by dilution of 6-HEX(AnaSpec) in activity buffer at concentrations ranging from 7 to 250 nMat 1.67-fold series dilutions. Fluorescence units were transformed toconcentration values (μM) by applying the calibration curve formula(y=ax+b when y=FU and a=nM). A double reciprocal plot of initialvelocity versus substrate concentration (at 2-15 μM) allows theextraction of K_(M) and V_(max) values by abscissa intercept with anasymptote to the high substrate region (FIG. 10B).

Specific Activity

Specific activity of human DNase I was evaluated using the DNaseAlert™fluorescence-quenched oligonucleotide probe (supplied by Ambion, cat#AM1970, manufactured by IDT) that emits a fluorescent signal uponnuclease degradation. One unit (U) of DNase is defined as the amount ofenzyme that releases 1 μmol of fluorescent product per minute at 22° C.The value of specific activity (U/mg) is calculated by dividing thevalue of total activity (U) by the amount of enzyme used in thereaction. Enzyme concentration was determined by absorbance at 280 nm,based on extinction coefficient. The calculated extinction coefficientof phr DNase I is 1.45 g/L.

2 nmol of DNaseAlert™ substrate (Ambion, cat #AM1970) was resuspendedwith 1 mL nuclease free water. Prh DNase I and Pulmozyme® samples werediluted 10,000-40,000-fold in activity buffer (25 mM HEPES-NaOH, 4 mMCaCl₂, 4 mM MgCl₂, 0.1% BSA, 0.05% Tween-20, pH 7.5). 80 uL of activitybuffer was divided in duplicates to a black 96-well plate (Greiner catNo. 655900). Ten microliters of substrate was then added and thereaction was initiated by rapid addition of 10 ul Rh DNase I orPulmozyme®. The plate was immediately incubated in a fluorometer andreal-time data was collected (535 nm excitation, 565 nm emission) at30-second intervals for 3 minutes. Product standard curve was preparedby dilution of 6-HEX (AnaSpec cat #81019) in activity buffer atconcentrations ranging from 7 to 250 nM at 1.67-fold series dilutions.Fluorescence units were transformed to concentration values (μM) byapplying the calibration curve formula (y=ax+b when y=FU and a=nM). 1 Uis defined as the amount of enzyme that releases 1 μmol of fluorescentproduct per minute at 22° C. The value of specific activity (U/mg) wascalculated by dividing the value of total activity (U) by the amount ofenzyme used in the reaction.

Results:

Plant Expressed rh DNase I has Superior Enzyme Kinetics Compared toMammalian Cell Expressed Human DNase I

FIG. 10A shows representative substrate inhibition kinetic plots ofplant expressed rh DNase I and Pulmozyme®. Initial velocity versussubstrate concentration plots yielded a curve that is typical for asubstrate inhibition pattern (see Table VI) for both enzymes. The K_(M),and V_(max) values were calculated from asymptote equation plotted in aregion of low substrate concentrations (2-15 μM) in double reciprocalplots (FIG. 10B). k_(cat) values of phr DNase I and Pulmozyme® werecalculated by dividing V_(max) with enzyme's concentration (Table VI),further indicating that prh DNase I and Pulmozyme® enzyme kineticsexhibit characteristics of the substrate inhibition kinetic model.

However, it is evident that although both enzymes comprise the sameDNase I amino acid sequence, prh DNase I exhibits improved kineticproperties (greater substrate affinity and greater enzyme velocity)compared to Pulmozyme®. Without wishing to adhere to a singlehypothesis, this may be due to unique post-translational modificationsof the plant-expressed rh DNase I generated as a result of expression ofthe nucleic acid construct of the present invention in plant, ratherthan mammalian expression systems.

Three batches of DNase extracted from cultured cells were analyzed andhave shown similar characteristics. Further analysis of batches of DNaseextracted from cells sampled from different lines of transformed BY2cells at different times confirmed the reproducible, favorable kineticcharacteristics of the plant expressed rh DNase I (see Table VIa below),as compared to Pulmozyme®.

TABLE VI Kinetic parameters of plant expressed rh DNase I vs.Pulmozyme ® K_(M) V_(max) k_(cat) k_(cat)/K_(M) DNase I (μM) (μM/min)(sec⁻¹) (sec⁻¹ μM⁻¹) Plant-expressed rh DNase I 24.3 0.58 6.24 0.26Pulmozyme ® 60.9 0.44 4.76 0.08

TABLE VIa Kinetic parameters of 3 plant expressed rh DNase I batchesprhDNase K_(M) V_(max) k_(cat) k_(cat)/K_(M) I line (μM) (μM/min)(sec⁻¹) (sec⁻¹ μM⁻¹) 1 16.77 0.37 5.58 0.33 2 12.54 0.36 5.44 0.43 212.07 0.35 5.29 0.44

Specific Activity of Plant-Expressed rh DNase I

Specific activity values of plant-expressed rh DNase I and mammaliancell expressed DNase I (Pulmozyme®) are presented in Table VII.Significantly, the specific activity of plant-expressed rh DNase I, asassayed with the DNaseAlert™ fluorescence-quenched oligonucleotide probeis about 3-fold higher than that of the Pulmozyme®. This difference inplant-expressed rh DNase I and Pulmozyme® specific activity reflects theimproved kinetic properties of plant-expressed rh DNase I compared toPulmozyme® (see FIGS. 10A and 10B and Table VI above).

TABLE VII Specific activity of plant-expressed rh DNase I vs. PulmozymeSpecific activity DNase I (U/mg) Plant-expressed rh DNase I 0.258 ±0.055 Pulmozyme ® 0.091 ± 0.018

Thus, plant-expressed prh DNase I is fully active catalytically, towardsconventional DNase I substrates, exhibits significantly superior enzymekinetics, and possesses a higher specific activity, consistent with theimproved enzyme kinetics, when compared to that of the commerciallyavailable, clinical standard mammalian cell expressed recombinant DNaseI.

Example 4 Superior Resistance of Plant-Expressed rh Dnase I to ActinInhibition

Analysis of the composition of CF sputum reveals large quantities (3 to14 mg/ml) of DNA and actin (0.06 to 5 mg/ml) released by necrosingneutrophils after their recruitment into the airways during response toinfection. In addition to hydrolysis of deoxynucleic acid, DNase I candepolymerize filamentous actin (F-actin). Monomeric globular actin(G-actin) is a potent inhibitor (Ki 1 nM) of DNase I enzymatic activity,potentially influencing the effectiveness of inhaled DNase I in CFlungs.

To evaluate G-actin inhibitory effect on DNase I activity, an IC₅₀ assay(half maximal inhibitory concentration) was developed, applying MethylGreen enzymatic activity assay in the presence of elevatedconcentrations of human non-muscle actin (Cytoskeleton; cat No. APHL99).

One hundred microliters of actin/DNase-I mixture was divided induplicates to a 96-well plate (NUNC cat No. 442404) containing 100 ul ofDNA-methyl green substrate. Human non-muscle actin (Cytoskeleton; catNo. APHL99) was diluted in activity buffer (25 mM HEPES-NaOH, 4 mMCaCl₂, 4 mM MgCl₂, 0.1% BSA, 0.05% Tween-20, pH 7.5) containing 0.1 mMATP (Sigma cat No. A26209) to reach concentrations ranging from 100 to0.1 ug/mL, at 2-fold series dilutions. Prh DNase I and Pulmozyme®(control) were diluted to reach concentration of 100 ng/mL. Each plate'scontent was then mixed thoroughly, plates were read at 620 nm, sealedand incubated for 4 hr at 37° C. and read again (620 nm). The change inabsorbance was plotted versus actin concentrations and IC₅₀ parameterswere calculated by non-linear fit using GraFit software (ErithacusSoftware, UK).

Results: Plant Expressed rh DNase I Exhibits Improved Resistance toActin Inhibition

FIG. 11 shows plots describing plant expressed rh DNase I and Pulmozyme®catalytic activity (expressed as ΔOD_(620 nm)) in the presence ofincreasing concentrations of human G-actin. The plot of the change inabsorbance versus actin concentration yields hyperbolic curves allowingthe extraction of IC₅₀ (Table VIII, below). Although both enzymes havethe same amino acid sequence, the plant expressed rh DNase I displaysgreater resistance to inhibition by human actin than that of thecommercial, mammalian cell expressed DNase (Pulmozyme®). Three batchesof DNase extracted from cultured cells were analyzed and have shownsimilar characteristics. Yet further, comparison ofresistance/susceptibility to actin inhibition of the plant expressedrhDNase I sampled from different lines of transformed BY2 cellsexpressing the recombinant enzyme (see Table VIIIa below) confirmed thereproducible improved resistance to actin inhibition.

Without wishing to be limited to a single hypothesis, one explanationfor this increased resistance to actin inhibition could be the result ofthe improved affinity of the plant expressed enzyme for its DNAsubstrate, as compared to that of the mammalian expressed enzyme, oractual reduced affinity of the prhDNase I for actin monomers, or both.

TABLE VIII IC₅₀ of plant expressed rh DNase I vs. Pulmozyme ® DNase IIC₅₀ (μg/ml) Prh DNase I 1.8191 ± 0.1003 Pulmozyme ® 0.6870 ± 0.0204

TABLE VIIIa IC₅₀ of plant expressed rh DNase I vs. Pulmozyme ® DNase IIC₅₀ (μg/ml) Prh DNase I batch 1 1.7067 ± 0.0568 Prh DNase I batch 21.8453 ± 0.0429 Pulmozyme ® 0.5458 ± 0.0115

Example 5 Effect of rh Dnase I on the Rheological Properties of Sputum

Mucus is defined as the heterogeneous, adhesive, viscoelastic gelproduced by goblet cells and submucosal glands. At the chemical level,mucus is an integrated structure of biopolymers. Its physical behavioris complex (non-Newtonian), with highly variable properties that arebetween those of a viscous liquid and an elastic solid.

Characterization of the physical properties of mucus largely focuses ontwo properties: (i) viscous modulus, also termed loss modulus (G″,expressed in pascal-seconds-Pa·s), which is the extent to which the gelresists the tendency to flow, and (ii) elastic modulus, also termedstorage modulus (G′, expressed in pascals-Pa seconds-Pa·s), whichexpresses the tendency for the gel to recover its original shapefollowing stress-induced deformation. Together, these propertiesdescribe the rheology of complex biological fluids. The phase angle orloss tangent value (δ), calculated from the inverse tangent of G″/G′(tan δ=G″/G′) is also a common parameter for characterizing mucus,reflecting the overall elastic or viscous nature of the sample and usedto quantify the extent of elastic behaviour of material. A phase angleclose to zero indicates strongly elastic behaviour, as opposed to 90°,which indicates purely viscous behaviour. A phase angle of 45° isconsidered the “cross over” value between G″ and G′.

CF Sputum Collection, Storage and Sample Treatment

CF sputum samples were collected from patients with severe lung diseaseattending Cystic Fibrosis Centers. Sputum was directly expectorated intoa sterile hermetically sealed container and transported on ice tolaboratories equipped for rheological characterization. Saliva wasremoved and each sputum sample was homogenized gently and divided into200-300 mg aliquots and stored at −70° C. until analyzed. Frozen sampleswere thawed at room temperature before analysis, as freezing of thesputum samples followed by a single thawing step was shown to affordaccurate and reproducible analysis of sputum rheology, similar to thoseof the fresh sample before freezing.

In order to ensure that the sputum is free of exogenous DNase I activity(e.g. Pulmozyme®), sputum samples were collected 12-24 hours aftertreatment with Pulmozyme® aerosol, as it has been reported that inhaledaerosol DNase I is cleared from the sputum from patients as soon as twohours.

Study Design

Each sputum aliquot was incubated for 30 min at 37° C. with DNase Iformulation buffer (1 mM CaCl₂, 150 mM NaCl, pH 6.1-6.5) containingeither prh DNase I or Pulmozyme® (final concentration 0.2, 2, 5, 10 and20 ug/gr sputum). Control samples were treated with DNase I formulationbuffer only.

Four percent (vol/wt) of drug or control were added to the sputumsample. Following incubation, rheological properties of sputum sampleswere immediately measured.

Sputum Rheology Assay

Rheological properties of the sputum samples were determined using acontrolled stress rheometer (HAAKE RheoStress 1, Thermo FisherScientific GmbH, Karlsruhe, Germany). The measurements were conducted at20° C. using two techniques: a time-sweep (FIGS. 12A-12D and 13A-13D)and a stress sweep (FIGS. 18A-18D and 19A-19D).

Time sweep measurements were performed using a 35 mm cone plate set-up.The angle between the cone and the plate was 0.5° and the sample volumerequired was 150 ul. Nondestructive oscillatory stress was applied tothe sample and the elastic (G′) and viscous (G″) modulus were recordedversus time. To avoid disruption of the weak biopolymer network in thesputum due to the oscillation forces, the measurements were performed inthe linear viscoelastic region at a constant frequency of 1 Hz with astress of 0.10 Pascals (Pa).

Stress sweep measurements were performed using 20 mm sandblastedparallel plate geometry. The sputum samples (200 ul) were loaded ontothe rheometer with a gap width of 0.5 mm A stress sweep was performedfrom 0.1 to 100 Pa at a constant frequency of 1 Hz and the elasticmodulus, (G′), viscous modulus (G″) and phase angle (δ) were measured.The applied stress in which G′ and G″ cross over (or phase angle reaches45°) is the stress in which the sample begins to act more liquid-likethan solid-like. At this point stress values were recorded and comparedbetween DNase I treated and untreated samples.

Rheological parameters were determined using RheoWin 4 (HAAKE, ThermoFisher Scientific GmbH, Karlsruhe, Germany). Before measurements, sputumsamples were loaded into the rheometer plate and equilibrated for 30seconds to allow relaxation to the original gel structure. In order toslow down the dehydration of the sputum, a solvent trap was used.Experiments were performed on at least two sputum fractions taken fromeach sputum sample and the data were averaged.

Measurements of Total DNA Content in Sputum

Sputum DNA content was determined by the modified aminobenzoic acid(DABA) assay. Salmon sperm DNA (Sigma #D1626, ˜2 mg/mL) and sputumsamples (˜100 mg) were diluted 10-fold in a dilution buffer (25 mMHEPES-NaOH, 4 mM CaCl₂, 4 mM MgCl₂, 0.1% BSA, 0.05% Tween-20, pH 7.5)and incubated at 60° C. for 1 hour. Samples were repeatedly vortexed toallow sputum disintegration. DNA concentration of the diluted salmonsperm sample was then measured (NanoDrop 2000, Thermo Fisher Scientific)and standard curve was prepared by dilution of the salmon sperm samplein the dilution buffer at concentrations ranging from 3.13 to 200 μg/mlat 2-fold series dilutions. Similarly, sputum samples were furtherdiluted at 2-fold series dilution up to 1280-fold. Next, fiftymicrolitres of standards and samples was added in duplicates to a black96-well plate (Greiner cat No. 655900) and incubated with 50 μL of 20%3,5-diaminobenzoic acid (TCI Europe cat No. D0079) at 60° C. for 1 hour.Fifty (50) ul of 5N HCl was then added to stop the reaction andfluorescence was measured by fluorometer (390 nm excitation/530 nmemission). Fluorescence units were plotted versus standard DNAconcentrations and the data were fit to a 4-parameter logistic model bythe nonlinear regression method of Marquardt. DNA concentration insputum was then interpolated.

Magnesium Chloride and rh DNase I Effect on Rheological Properties ofSputum

Magnesium ion is a cofactor of DNase I, hence promoting DNA hydrolysis.Studies suggest that increasing magnesium concentration in the airwaysurface liquid by aerosolisation of magnesium solutions or oralmagnesium supplements could improve the removal of highly viscous mucusin chronic lung disease by improving DNase activity. Furthermore, it wassuggested that magnesium indirectly triggers the activity of rhDNase Iin CF sputum by promoting polymerization of G-actin into F-actin, hence,reducing inhibition of DNase-I by G-actin. Consequently, magnesiumsupplement may overcome poor response to rhDNase treatment innon-responding CF patients exhibiting low levels of magnesium in theirsputum.

Magnesium Sulfate and rh DNase I Effect on Catalytic Activity of rhDNase I

Several trials conducted with inhaled magnesium sulfate in patients withacute asthma have shown that magnesium sulfate inhalation is tolerable.Magnesium ions, required for DNase I activity, can therefore besupplemented to rh DNase I formulation as magnesium sulfate. To verifythat magnesium sulfate does not produce an inhibitory effect on rh DNaseI activity a methyl green activity assay was applied in the presence ofelevated concentrations of magnesium sulfate and compared to magnesiumchloride effect.

One hundred microliters of plant expressed rh DNase I was divided induplicates to a 96-well plate (NUNC cat No. 442404) containing 100 ul ofDNA-methyl green substrate to reach concentration of 100 ng/mL. Reactionwas prepared in a modified activity buffer (25 mM HEPES-NaOH, 4 mMCaCl₂, 0.1% BSA, 0.05% Tween-20, pH 7.5) containing elevatedconcentration of magnesium sulfate or magnesium chloride (0.5-100 mM).The plate's contents were then mixed thoroughly, and the plates wereread at 620 nm, sealed and incubated for 3 hr at 37° C. and re-read (620nm). Change in absorbance was plotted versus concentrations of magnesiumsulfate or magnesium chloride added.

Results:

Plant Expressed rh DNase I is Highly Effective in Reducing RheologicalProperties of Sputum of CF Patients.

The rheological properties of sputum samples were measured followingincubation of CF sputum with DNase I formulation buffer or differentconcentrations of plant expressed rh DNase I and Pulmozyme®, diluted inDNase I formulation buffer (final concentration 0.2, 2, 5, 10 and 20ug/gr sputum) (see FIGS. 12A-12D; 13A-13D; 18A-18D and 19A-19D). DNase Iconcentrations correspond to the concentrations detected 15 min afteraerosolization of Pulmozyme® recommended therapeutic dose (mean value of2.9 μg/ml mucus).

Time sweep measurement (FIGS. 12A-12D and 13A-13D) clearly revealed thatincubation with the plant expressed rh DNase I significantly reducedsputum elastic modulus (FIG. 12A-12D) and viscous modulus (FIG.13A-13D), in a concentration-dependent manner, and with a consistentlygreater efficiency (per ug DNase I; FIG. 12A) than the clinicallyapproved mammalian cell-expressed Pulmozyme®. Even greater expression ofthe improved rheological properties of the prh DNase I-treated sputa wasobserved when the reduction elastic and viscous modulus followingincubation with prh DNase I was expressed as percent change compared tothe untreated control (see FIGS. 14A and 14B). In addition, stress sweepmeasurements (see FIGS. 18A-18D and 19A-19D) reveals that the plantexpressed rh DNase I disrupts the sputa elastic structure (the internalnetwork of the sputa) in a dose-dependent manner and with greaterefficiency than the clinically approved mammalian cell-expressedPulmozyme®. The improved efficacy of the prh DNase I is consistent withthe improved enzyme kinetics and the higher specific activity of the prhDNase I, compared to Pulmozyme® (see Examples 2 and 3 above).

Synergistic Reduction of CF Sputum Rheological Properties with PlantExpressed rh DNase I and Magnesium

Time sweep measurements evaluating the rheological properties of CFsputum were performed following incubation of sputum samples with plantexpressed rh DNase I protein, diluted in DNase I formulation buffer(final concentration of 2 ug/gr sputum), or DNase I formulation buffer(control), in the presence or absence of 25, 50 and 100 mM magnesiumchloride, prepared in DNase I formulation buffer. FIGS. 15 and 16clearly indicate that incubation of sputum with 25-100 mM magnesiumchloride (grey bars), or prhDNase alone (“0” MgCl₂ in FIGS. 15 and 16)results in reduction in both the sputum elastic (FIG. 15) and viscous(FIG. 16) modulus. When the sputum is incubated with both magnesiumchloride and plant expressed rh DNase I, (hatched bars, 25-100 mMMgCl₂), a significant synergistic reduction can be observed following athreshold of added Mg (in this experiment 100 mM Mg) in both sputumrheological properties assayed [elastic (FIG. 15) and viscous (FIG. 16)modulus].

Stress sweep measurements were performed following incubation of sputumsamples with plant expressed rh DNase I protein, diluted in DNase Iformulation buffer, or DNase I formulation buffer (control), in thepresence or absence of 100 mM magnesium sulfate, prepared in DNase Iformulation buffer (see FIGS. 20A-20C). Taken together, these resultsdemonstrate that incubation of CF sputum with both magnesium sulfate(FIG. 20A-20C, grey bars) and prhDNase (FIG. 20A-20C, dark bars) resultsin a synergistically greater and more significant disruption in sputumelastic structure than incubation with prhDNase or magnesium sulfatealone. This is particularly evident in patient sample A, FIG. 20A, wherereduction in the stress sweep values for sputum sample by prhDNase wasdetected primarily when magnesium sulfate is added.

Magnesium Salts do not Inhibit Plant Expressed rh DNase I Activity

When catalytic activity of plant expressed rh DNase I was assayed in thepresence of different concentrations of magnesium sulfate and magnesiumchloride, a dose-dependent improvement of DNA hydrolytic activity wasobserved with concentrations between 0.5 to 10 mM magnesium, with nodifference between activity in the presence of chloride salt (MgCl₂) orsulphate salt (MgSO₄) (FIG. 16). DNA hydrolytic activity of the plantexpressed rh DNase I remained optimal with both magnesium sulfate andmagnesium chloride at concentration of 10-50 mM, with only a slightdepression of activity with magnesium sulphate, compared to magnesiumchloride, at 100 mM. Thus, neither the chloride salt (MgCl₂) or sulphatesalt (MgSO₄) significantly impairs DNA hydrolytic activity of the plantexpressed rh DNase I over a wide range of concentrations.

Taken together, these results show that incubation with plant expressedrh DNase I improves the rheological properties of CF sputum. The dosedependent reduction in sputum rheological properties achieved with prhDNase I is more dramatic than that observed with mammalian cellexpressed DNae I (Pulmozyme®). Yet further, it was observed thataddition of magnesium chloride can improve the effect of plant expressedrh DNase I on the rheological properties of sputum, and that the DNAhydrolytic activity of plant expressed rh DNase I is enhanced by bothmagnesium chloride and magnesium sulphate over a wide range ofconcentrations. Thus, magnesium sulfate can be supplemented to prh DNaseI to improve the prh DNase I's mucolytic activity in-vivo.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

1. An inhalable dry powder formulation comprising a human DNase Iprotein and particles of a physiologically acceptablepharmacologically-inert solid carrier.
 2. The inhalable dry powderformulation of claim 1, wherein said human DNase I protein is arecombinant plant-expressed human DNase I protein.
 3. (canceled)
 4. Theinhalable dry powder of claim 2, wherein said human DNase I proteincomprises an N-terminal Glycine residue.
 5. The inhalable dry powderformulation of claim 1, wherein said human DNase I protein comprises theamino acid sequence as set forth in SEQ ID NO:
 6. 6. The inhalable drypowder formulation of claim 4, wherein said human DNase I proteincomprises the amino acid sequence as set forth in SEQ ID NO:5.
 7. Theinhalable dry powder formulation of claim 1, wherein said human DNase Iprotein has at least one core xylose and at least one core α-(1,3)fucose.
 8. The inhalable dry powder formulation of claim 2, wherein saidhuman DNase I protein has reduced susceptibility to actin inhibition ofendonuclease activity as compared with that of mammalian cell expressedhuman recombinant DNase I.
 9. The inhalable dry powder of claim 1,wherein said carrier is selected from the group consisting of (a) atleast one crystalline sugar selected from the group consisting ofglucose, arabinose, maltose, saccharose, dextrose, and lactose; and (b)at least one polyalcohol selected from the group consisting of mannitol,maltitol, lactitol, and sorbitol.
 10. The inhalable dry powderformulation of claim 1, wherein said carrier is in a form of finelydivided particles having a mass median diameter (MMD) in the range of0.5 to 10 microns.
 11. (canceled)
 12. The inhalable dry powderformulation of claim 1, wherein said carrier is in a form of coarseparticles having a mass diameter of 50-500 microns.
 13. (canceled) 14.The inhalable dry powder formulation of claim 1, wherein said carriercomprises a mixture of coarse particles having a mass diameter of 150microns to 400 micron and finely divided particles having a MMD in therange of 0.5-10 microns.
 15. The inhalable dry powder formulation ofclaim 1, further comprising, as an active ingredient, a magnesium salt.16. The inhalable dry powder formulation of claim 1, further comprising,as an active ingredient, an agent for inhibiting formation of G actinand/or enhancing formation of F actin.
 17. (canceled)
 18. The inhalabledry powder formulation of claim 1, wherein said human DNase I is inassociation with diketopiperazine.
 19. (canceled)
 20. The inhalable drypowder formulation of claim 1, wherein said human DNase I protein is atleast 90-95% pure human DNase I protein.
 21. The inhalable dry powderformulation of claim 1, further comprising plantbeta-acetylhexosaminidase enzyme protein.
 22. The inhalable dry powderformulation of claim 21, wherein said plant beta-acetylhexosaminidaseenzyme protein is inactivated beta-acetylhexosaminidase enzyme protein.23. A dry powder inhaler device, comprising the inhalable dry powderformulation of claim 1 and a means for introducing the inhalable drypowder formulation into the airways of a subject by inhalation. 24-26.(canceled)
 27. The dry powder inhaler device of claim 23, wherein saidsubject is suffering from a disease or condition selected from the groupconsisting of male infertility, metastatic cancer, a viral, bacterial,fungal or protozoan infection, sepsis, atherosclerosis, diabetes,delayed type hypersensitivity and a uterine disorder.
 28. A method ofreducing DNA in a pulmonary secretion or fluid of a subject in needthereof, the method comprising administering to the subject theinhalable dry powder formulation of claim 1, thereby reducing DNA in thepulmonary secretion or fluid of the subject.
 29. A method of preventingand/or treating a pulmonary disease or condition associated with excessDNA in a pulmonary secretion in a subject in need thereof, the methodcomprising administering to the subject the inhalable dry powderformulation of claim 1, thereby preventing and/or treating the pulmonarydisease or condition associated with excess DNA in the pulmonarysecretion in the subject.
 30. The method of claim 28, wherein saidsubject is suffering from a respiratory disease selected from the groupconsisting of acute or chronic bronchopulmonary disease, atelectasis dueto tracheal or bronchial impaction, and complications of tracheostomy.31. The method of claim 30, wherein said acute or chronicbronchopulmonary disease is selected from the group consisting ofinfectious pneumonia, bronchitis or tracheobronchitis, bronchiectasis,cystic fibrosis, asthma, chronic obstructive pulmonary disease (COPD),TB or fungal infections. 32-36. (canceled)
 37. The method of claim 31,wherein an effective amount of said dry powder formulation is aplurality of doses, each dose comprising 1.0-3.0 mg DNase, said dosesadministered at least twice, 2-3 times, 2-4 times or 2-6 times daily.38-39. (canceled)